Peripheral Leukocytes Research Articles

ANALYSIS OF IMMUNE CELLS AND IMMUNOLOGICAL PROCESSES IN COVID-19

The given scientific article analyses the dynamics of quantitative alterations in peripheral leukocytes and hematopoietic elements in blood during the period of COVID-19 and recovery. We studied the effect of SARS-CoV-2 infection on the hematopoietic system, lymphocytopenia and eosinopenia during the course of the disease, control of the recovery period from thedisease by monitoring the dynamics of the number of formed elements.

Single-nucleus transcriptome unveils the role of ferroptosis in ischemic stroke

Multiple cell death pathways are involved in neuronal death in ischemic stroke (IS). However, the role of different cell death pathways in different cell types has not been elucidated. By analyzing three single-nucleus RNA sequencing (snRNA-seq) data of IS, we first found that a variety of programmed cell death (PCD) -related genes were significantly changed in different cell types. Based on machine learning and virtual gene knockout, we found that ferroptosis related genes, ferritin heavy chain 1 (Fth1) and ferritin light chain (Ftl1), play a key role in IS. Ftl1 and Fth1 can promote microglia activation, as well as the production of inflammatory factors and chemokines. Cell communication analysis showed that activated microglia could enhance chemotactic peripheral leukocyte infiltration, such as macrophages and neutrophils, through Spp1-Cd44 and App-Cd74 signaling, thereby aggravating brain tissue damage. Furthermore, real‐time quantitative polymerase chain reaction (RT‐qPCR) showed that P2ry12 and Mef2c were significantly decreased in oxygen-glucose deprivation (OGD) group, while Ftl1, Fth1, Apoe, Ctsb, Cd44 and Cd74 were significantly increased in OGD group. Collectively, our findings suggested targeted therapy against microglia Ftl1 and Fth1 might improve the state of microglia, reduce the infiltration of peripheral immune cells and tissue inflammation, and then improve the ischemic brain injury in mouse.

Cerebroprotective Effects of the TLR4-Binding DNA Aptamer ApTOLL in a Rat Model of Ischemic Stroke and Thrombectomy Recanalization.

ApTOLL, a TLR4 modulator aptamer, has demonstrated cerebroprotective effects in a permanent ischemic stroke mouse model, as well as safety and efficacy in early phase clinical trials. We carried out reverse translation research according to STAIR recommendations to further characterize the effects and mechanisms of ApTOLL after transient ischemic stroke in rats and to better inform the design of pivotal clinical trials. Adult male rats subjected to transient middle cerebral artery occlusion were treated either with ApTOLL or the vehicle intravenously at different doses and time-points. ApTOLL was compared with TAK-242 (a TLR4 inhibitor). Female rats were also studied. After neurofunctional evaluation, brains were removed for infarct/edema volume, hemorrhagic transformation, and histologic determinations. Peripheral leukocyte populations were assessed via flow cytometry. ApTOLL showed U-shaped dose-dependent cerebroprotective effects. The maximum effective dose (0.45 mg/kg) was cerebroprotective when given both before reperfusion and up to 12 h after reperfusion and reduced the hemorrhagic risk. Similar effects occurred in female rats. Both research and clinical ApTOLL batches induced slightly superior cerebroprotection when compared with TAK-242. Finally, ApTOLL modulated circulating leukocyte levels, reached the brain ischemic tissue to bind resident and infiltrated cell types, and reduced the neutrophil density. These results show the cerebroprotective effects of ApTOLL in ischemic stroke by reducing the infarct/edema volume, neurofunctional impairment, and hemorrhagic risk, as well as the peripheral and local immune response. They provide information about ApTOLL dose effects and its therapeutic time window and target population, as well as its mode of action, which should be considered in the design of pivotal clinical trials.

#1864 Phosphorilation of SHP-1 switch does not indicate lupus nephritis activity

Abstract Background and Aims Protein tyrosine kinases (PTKs) are enzymes responsible for the phosphorylation of tyrosine residues in lymphocytes. In 2017, Mkaddem et al. suggested that unique PTK signatures in peripheral leukocytes (PBMC) are associated with lupus nephritis (LN) activity [1]. Lymphocytes express two important PTKs, Lyn and Fyn, that modulate inhibitory or activatory signals depending on ligand avidity or phosphorylation status. SHP-1 tyrosine 536 phosphorylation (pSHP1-Y536) by Lyn activates inhibitory signaling and Fyn-dependent phosphorylation of SHP-1 serine 591 (pSHP1-S591) inactivates the phosphatase, enabling activatory immunoreceptor signaling. We hypothesized that active and refractory patients with proliferative LN have a different PTK profile in PBMC, with lower expression of pSHP1-Y536 and greater expression of pSHP1-S591, in comparison with those who have responded to treatment. Method In a cohort from the Royal Free Hospital, UCL (London, UK) we performed western blotting of PBMC lysates for SHP1-phospho-S591 and-Y536 from healthy individuals (HC; n = 19), LN patients in remission (n = 11) and with ongoing disease activity (n = 4). Densitometry of bands was carried out and a ratio of S591:Y536 expression was calculated (both normalized to actin). For the flow cytometry analysis, we followed up a Brazilian cohort of LN (n = 24) from Hospital das Clinicas, UFPE (Recife, Brazil). All patients had ongoing active LN with initial renal biopsy (class III, IV or/and V). PBMC samples were collected at 0, 3, 6, 9, 12, 18, and 24 months after biopsy. Isolated PBMC samples were stored at −80°C. PBMC samples were stained with antibodies against pSHP1 S591 and p-SHP1 Y536 and cell viability was checked using zombie red dye. Samples were acquired on the Accuri C6 cytometer (BD) and analyzed using the FLOJO software (BD). Results The western blotting relative expression of pSHP-1 S591:Y536 in PBMC was not significantly different when we compared HC with active LN (p = 0.098) and active with remission LN (p = 0.10), Fig. 1. The results obtained by flow cytometry expression ratio of pSHP1-S591:Y536 did not show a significant correlation with the median of proteinuria (Fig. 2), C3, C4, or SLEDAI from time of active kidney biopsy during LN flare throughout induction treatment. Conclusion There was no expression pattern of pSHP-1 Y536 and S591 in PBMC of LN patients, regardless of their renal activity or inflammatory status. Therefore, we cannot state those markers as a potential “liquid biopsy” for LN.

EPAS1-related pheochromocytoma/paraganglioma.

In 2012, somatic EPAS1 pathogenic variants were found to cause a triad of pheochromocytoma/paragangliomas (PPGLs), polycythemia, and somatostatinoma. Since then, a limited number of studies on this subject have been reported, and data on the long-term outcome of metastatic disease are not available on this rare syndrome. We comprehensively reviewed EPAS1-related PPGL and describe an unusual patient who has been living with an EPAS1-related metastatic PPGL for 47 years. The results of this work show that EPAS1 pathogenic variants are rare, more in females and patients without pathogenic variants in other PPGL susceptibility genes. PPGLs are the most common manifestation followed by polycythemia and somatostatinoma. The EPAS1 pathogenic variants are often postzygotic, and the timing of their acquirement during embryonic development seems to correlate with the number and timing of development of the disease manifestations. Although recurrent and multifocal disease is common in EPAS1-related PPGL, distant metastases are uncommon and usually indolent. This is illustrated by a case of a man who was diagnosed at the age of 9 years and is currently 56 years old, alive, and well for 47 years with these metastases. He was found to have a somatic EPAS1 pathogenic variant (c.1592C>A, p.Pro531His) in bilateral pheochomocytoma and a pancreatic NET (somatostatinoma) but not in genomic DNA isolated from peripheral leukocytes. This and previous reports suggest that distant metastases are uncommon and less aggressive in EPAS1-related PPGLs compared to those found in other hereditary PPGLs.

Identification of circRNA Expression Profile and Potential Systemic Immune Imbalance Modulation in Premature Rupture of Membranes.

Premature rupture of membrane (PROM) refers to the rupture of membranes before the onset of labor which increases the risk of perinatal morbidity and mortality. Recently, circular RNAs (circRNAs) have emerged as promising regulators of diverse diseases. However, the circRNA expression profiles and potential circRNA-miRNA-mRNA regulatory mechanisms in PROM remain enigmatic. In this study, we displayed the expression profiles of circRNAs and mRNAs in plasma and fetal membranes of PROM and normal control (NC) groups based on circRNA microarray, the Gene Expression Omnibus database, and NCBI's Sequence Read Archive. A total of 1,459 differentially expressed circRNAs (DECs) in PROM were identified, with 406 upregulated and 1,053 downregulated. Then, we constructed the circRNA-miRNA-mRNA network in PROM, encompassing 22 circRNA-miRNA pairs and 128 miRNA-mRNA pairs. Based on the analysis of gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene set enrichment analysis (GSEA), DECs were implicated in immune-related pathways, with certain alterations persisting even postpartum. Notably, 11 host genes shared by DECs of fetal membrane tissue and prenatal plasma in PROM were significantly implicated in inflammatory processes and extracellular matrix regulation. Our results suggest that structurally stable circRNAs may predispose to PROM by mediating systemic immune imbalances, including peripheral leukocyte disorganization, local immune imbalance at the maternal-fetal interface, and local collagen disruption. This is the first time to decipher a landscape on circRNAs of PROM, reveals the pathogenic cause of PROM from the perspective of circRNA, and opens up a new direction for the diagnosis and treatment of PROM.

Greater methylation of the IL-6 promoter region is associated with decreased integrity of the corpus callosum in schizophrenia

BackgroundSchizophrenia is associated with chronic subclinical inflammation and decreased integrity of the corpus callosum (CC). Our previous study showed associations between peripheral IL-6 levels and the integrity of the CC. Epigenetic studies show associations between methylation of the genes related to immunological processes and integrity of the CC. AimTo investigate correlations between methylation status of IL-6 promotor and peripheral IL-6 levels and the integrity of the CC in schizophrenia. Material and methodsThe participants were 29 chronic schizophrenia patients (SCH) and 29 controls. Decreased integrity of the CC was understood as increased mean diffusivity (MD) and/or decreased fractional anisotropy (FA) in diffusion tensor imaging. Peripheral IL-6 concentrations were measured in serum samples and IL-6 promoter methylation status of 6 CpG sites was analyzed in peripheral leukocytes by pyrosequencing. ResultsModerate positive correlations were found between CpG1 methylation and the MD of proximal regions of the CC (CCR1–CCR3) and between CpGmean and MD of CCR1 in SCH. Weaker positive correlations were found for CpGmean with CCR2 and CCR3 and negative correlations were found for CpG1 and FA of CCR3 in SCH. Multivariate regression showed that methylation of CpG1, type of antipsychotic treatment, and their interaction were significant independent predictors of MD of CCR1 in SCH. Methylation of CpG2 was negatively correlated with serum IL-6 in SCH. ConclusionsThe methylation level of the IL-6 promotor region in peripheral leukocytes is associated with the integrity of the CC in schizophrenia and this association may depend on the type of antipsychotic treatment. Further studies are necessary to explain the mechanisms of the observed associations.

Abstract PO4-12-08: Changes in peripheral immune cell composition in women who do and do not develop breast cancer

Abstract Background: Breast cancer survivors have higher age-specific rates of hypertension and other chronic diseases than women without a history of breast cancer. We and others have shown that alterations to the immune system are associated with risk of these conditions in the general population, leading to the hypothesis that the increased chronic disease risk in breast cancer survivors may be driven, in part, by lasting changes in immunity. Peripheral immune cell composition appears to become altered years before a breast cancer diagnosis, but little is currently known about the influence of different breast cancer treatments on subsequent changes to leukocyte composition and the persistence of these associations over time. Methods: Among 410 women enrolled in the Sister Study, paired blood samples collected an average of 7.6 years apart were analyzed for DNA methylation (DNAm). Deconvolution methods were applied to these DNAm data to estimate circulating percentages of 12 leukocyte subsets. Approximately half the women sampled were diagnosed and treated for breast cancer between the blood draws (n= 185) whereas the other half remained breast cancer-free (n= 225). Breast tumor characteristics and treatment information were abstracted from medical records. Mixed-effect linear regression models were used to estimate changes in leukocyte composition over time comparing women with breast cancer to those who remained breast cancer-free. A case-only analysis of breast cancer survivors was performed to examine the persistence of changes over time and to explore whether changes in leukocyte composition were associated with the types of therapies received (endocrine therapy, radiation therapy, chemotherapy). All models were adjusted for age and self-reported race. In the treatment analysis, because tumor characteristics can guide clinical decisions, tumor estrogen receptor status and stage at diagnosis were additionally included as model covariates. Results: At baseline, women who developed breast cancer between the blood draws had lower mean circulating percentages of CD8+ cytotoxic T cells than women who remained breast cancer-free (3.8% vs 4.6%; P-diff= 0.04). After accounting for differences in leukocyte composition at baseline, compared to women who remained breast cancer-free, women diagnosed and treated for breast cancer between the blood draws had decreases in total circulating CD4+ helper T cell percentage (adjusted mean difference [β]= -1.50, 95% CI: -2.56, -0.44, P= 0.006) and alterations to both naïve and memory B cell percentages (naïve B cells, β= 0.46, 95% CI: 0.17, 0.75, P= 0.002; memory B cells, β= -0.22, 95% CI: -0.34, -0.09, P= 0.001). Although associations did not vary by tumor characteristics or participant race, the changes in leukocyte composition appeared to persist over time as changes were not associated with time since diagnosis. In the case-only analysis of different breast cancer therapies, radiation was associated with decreases in CD4+ T cells (β= -2.56, 95% CI: -4.26, -0.88, P= 0.003) and chemotherapy was associated with increases in B cells (β= 0.62, 95% CI: 0.07, 1.16, P= 0.03). Surgery and endocrine therapy were not meaningfully associated with changes in leukocyte composition. Conclusions: Breast cancer survivors have lasting changes in peripheral leukocyte composition that may be related to the types of treatments received. These findings add to our understanding of the biological changes that underlie the long-term health of breast cancer survivors. Citation Format: Jacob Kresovich, Katie O'Brien, Zongli Xu, Clarice Weinberg, Dale Sandler, Jack Taylor. Changes in peripheral immune cell composition in women who do and do not develop breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-12-08.

Repeated allergen consumption impairs intestinal barriers and promotes neurobehavioral pathologies in mice with subclinical cow’s milk allergy

Abstract Cow’s milk and other food allergens are often implicated in behavioral problems in sensitized but tolerant individuals. While the mechanisms are still unclear, we previously showed that male C57BL/6J mice with subclinical cow’s milk allergy (CMA) exhibited depression-like behavior associated with brain neuroinflammation and cortical demyelination after consuming a whey protein-containing diet (WP diet) for 2 weeks. Recently, we also found that adoptively transferred peripheral leukocytes from CMA mice migrated to the dura mater of naïve recipient mice during the duration of the 2-week WP diet. Hypothesizing that consumption of food allergens by subclinically sensitized individuals would cause intestinal damage and promote the trafficking of activated peripheral leukocytes to the dura to adversely affect the brain, we assessed neurobehavioral and intestinal pathologies CMA mice that sensitized to β-lactoglobulin (Bos d 5) and exposed to WP diet for 2 weeks. Behavioral tests indicated that the exploratory activity and short-term memory of CMA mice were significantly lower than controls. Cortical demyelination, microgliosis, and axonal damage were detected in the brain as previously reported. Notably, immunoreactivity for a tight junction protein, claudin 5, was visibly lower in the intestines of CMA mice. Our results suggested that repeated allergen consumption could trigger immune responses by increasing gut permeability in susceptible individuals.

Disordered GPR43/NLRP3 expression in peripheral leukocytes of patients with atrial fibrillation is associated with intestinal short chain fatty acids levels

BackgroundAtrial fibrillation (AF) is associated with circulating inflammation. Short-chain fatty acids (SCFAs) derived from gut microbiota (GM) regulate leukocyte function and inhibit the release of inflammatory cytokines, which are partly mediated by the G-protein-coupled receptor 43 (GPR43) signaling. This study aimed to investigate the expression of GPR43/NOD-like receptors family pyrin domain containing 3 (NLRP3) in leukocytes and the interaction with intestinal SCFAs levels in AF patients.MethodsExpressions of GPR43 and NLRP3 mRNA in peripheral blood leukocytes from 23 AF patients and 25 non-AF controls were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expressions of leukocyte GPR43 and NLRP3 protein were evaluated by western blot analysis. The levels of plasma IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). The fecal SCFAs levels based on GC/MS metabolome of corresponding 21 controls and 14 AF patients were acquired from our published dataset. To evaluate the expression of NLRP3 and GPR43 and the release of IL-1β, human THP-1 cells were stimulated with or without SCFAs (acetate, propionate, and butyrate), lipopolysaccharide (LPS), and nigericin in vitro, respectively.ResultsCompared to the controls, the mRNA expression in peripheral leukocytes was significantly reduced in AF patients (P = 0.011) coupled with the increase in downstream leukocyte NLRP3 mRNA expression (P = 0.007) and plasma IL-1β levels (P < 0.001), consistent with changes in GPR43 and NLRP3 protein expression. Furthermore, leukocyte GPR43 mRNA levels were positively correlated with fecal GM-derived acetic acid (P = 0.046) and negatively correlated with NLRP3 mRNA expression (P = 0.024). In contrast to the negative correlation between left atrial diameter (LAD) and GPR43 (P = 0.008), LAD was positively correlated with the leukocyte NLRP3 mRNA levels (P = 0.024). Subsequent mediation analysis showed that 68.88% of the total effect of intestinal acetic acid on AF might be mediated by leukocyte GPR43/NLRP3. The constructed GPR43–NLRP3 score might have a predictive potential for AF detection (AUC = 0.81, P < 0.001). Moreover, SCFAs treatment increased GPR43 expression and remarkably reduced LPS/nigericin-induced NLRP3 expression and IL-1β release in human THP-1 cells in vitro.ConclusionsDisrupted interactions between GPR43 and NLRP3 expression in peripheral blood leukocytes, associated with reduced intestinal GM-derived SCFAs, especially acetic acid, may be involved in AF development and left atrial enlargement by enhancing circulating inflammation.

Safety profile and effects on the peripheral immune response of fecal microbiota transplantation in clinically healthy dogs.

Fecal microbiota transplantation (FMT) is increasingly used for gastrointestinal and extra-gastrointestinal diseases in veterinary medicine. However, its effects on immune responses and possible adverse events have not been systematically investigated. Determine the short-term safety profile and changes in the peripheral immune system after a single FMT administration in healthy dogs. Ten client-owned, clinically healthy dogs as FMT recipients, and 2 client-owned clinically healthy dogs as FMT donors. Prospective non-randomized clinical trial. A single rectal enema of 5 g/kg was given to clinically healthy canine recipients. During the 28 days after FMT administration, owners self-reported adverse events and fecal scores. On Days 0 (baseline), 1, 4, 10, and 28 after FMT, fecal and blood samples were collected. The canine fecal dysbiosis index (DI) was calculated using qPCR. No significant changes were found in the following variables: CBC, serum biochemistry, C-reactive protein, serum cytokines (interleukins [IL]-2, -6, -8, tumor necrosis factor [TNF]-α), peripheral leukocytes (B cells, T cells, cluster of differentiation [CD]4+ T cells, CD8+ T cells, T regulatory cells), and the canine DI. Mild vomiting (n = 3), diarrhea (n = 4), decreased activity (n = 2), and inappetence (n = 1) were reported, and resolved without intervention. Fecal microbiota transplantation did not significantly alter the evaluated variables and recipients experienced minimal adverse events associated with FMT administration. Fecal microbiota transplantation was not associated with serious adverse events, changes in peripheral immunologic variables, or the canine DI in the short-term.

Expression of hTERT and Lp-PLA(2) genes is increased in primary hypothyroidism.

ObjectiveSightly low serum free T4 levels are associated with longer life expectancy. However, hypothyroidism is associated with increased risk of inflammation and vascular complications. Therefore, relation of thyroid hormones (THs) with life expectancy seems to be complex. This study was carried out in an effort to understand the contribution of THs in inflammation and aging. Methodology15 cases of treatment naïve primary hypothyroidism and 15 age and sex matched euthyroid controls were recruited. mRNA expression of Telomerase (hTERT), leucocyte telomere length (LTL) and Lipoprotein associated phospholipase A2 (Lp-PLA2), a marker of vascular inflammation and risk predictor of cardiovascular events were measured by qPCR in whole blood. ResultExpression of hTERT was found to be 6.95 times higher in the cases as compared to the controls. mRNA expression of Lp-PLA(2) was also 3.3 times higher in cases. LTL in euthyroid was approximately 81% of the length in hypothyroid patients. ConclusionA higher expression of hTERT indicates that hypothyroidism confers better cell survival in peripheral leucocytes inspite of the presence of vascular inflammation. However causal relationship cannot be ascertained with these results.

Abstract 3461: Shorter leukocyte telomere length and higher risk of primary liver cancer in a prospective cohort study

Abstract Background: Telomeres are conserved tandem repeats at chromosome ends that are vital to maintaining chromosomal stability and preventing degradation. Shorter telomere lengths (TL) are typically associated with older biological age or poor health status and can activate programmed cell death, whereas longer telomere lengths can be indicative of younger age or abnormal replication processes with oncogenic mutations. Previous research has identified a link between telomere length and carcinogenesis, but relatively few studies have investigated TL and primary liver cancer (PLC) risk despite it being one of the leading causes of cancer worldwide. The goal of this analysis was to study this association in a large prospective cohort. Methods: Data was extracted from the Singapore Chinese Cohort Health Study, a cohort of 63,257 men and women aged 45-74 years at enrollment during 1993-1998. Telomere length was measured in peripheral leukocytes of 26,540 participants who provided baseline blood samples, using a validated quantitative polymerase chain reaction (qPCR) method. PLC incidence and death among cohort participants was annually surveilled using the nationwide Singapore Cancer Registry and the National Birth and Death Registry from study enrollment to 2015. For statistical analysis, a t test was performed to examine the differences in TL between participants who developed incident PLC and those who remained free of PLC. Cox proportional hazard method was used to estimate the hazard ratios (HRs) and their 95% confidence interval (CI) of PLC incidence according to TL quartiles. Results: Over a 12-year follow-up, 199 participants developed incident PLC. The relative TL was significantly lower in participants with incident PLC than those without (-6.7%, p &amp;lt; 0.001). Multivariate Cox regression model showed an inverse association between TL quartile and risk of PLC (p for trend = 0.009). Compared with the lowest quartile, HRs (95% CIs) of PLC for the 2nd, 3rd, and highest quartile of TL were 0.78 (0.54-1.12), 0.75 (0.51-1.11), and 0.57 (0.37-0.88), respectively, after adjustment for age, sex, alcohol use, body mass index, and smoking status. Conclusion: The present analysis demonstrated a statistically significant association between shorter telomere length and increased risk of PLC in a large prospective cohort of Singaporean Chinese. This inverse association for PLC is different from those for other cancer types in the same cohort. These results warrant further studies such as using Mendelian randomization to quantify genetic variation in TL with PLC development. Citation Format: Sindhu Karnam, Kathryn Demanelis, Kenneth B. Beckman, Renwei Wang, Jennifer Adams-Haduch, Woon-Puay Koh, Jian-Min Yuan. Shorter leukocyte telomere length and higher risk of primary liver cancer in a prospective cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3461.

Implications of the Matrix Metalloproteinases, Their Tissue Inhibitors and Some Other Inflammatory Mediators Expression Levels in Children Obesity-Related Phenotypes.

Matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing endo-peptidases engaged in many biological processes including adipogenesis, angiogenesis, and tissue remodeling. Fat tissue infiltration by peripheral leukocytes plays an important role in transition of fat tissue residual, non-inflammatory status into the pro-inflammatory one, resulting in fat tissue inflammation and expansion as well as production of many mediators like adipokines and cytokines. The aim of this study was to investigate the expression of MMPs, their endogenous tissue inhibitors (TIMPs), and selected inflammatory mediators in leukocytes and plasma of children with simple obesity to find their associations with obesity-related phenotypes. Twenty-six overweight/obese children and twenty-three healthy volunteers participated in the study. The leukocyte mRNA expression levels of MMP-2, -9, -12 -14, TIMP-1, -2, and IL-6 were analyzed by the real time quantitative PCR. Plasma MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios as well as the concentrations of MMP-9, TIMP-1, IL-1 beta, IL-6, TNF- alpha, leptin and resistin were tested by ELISA assays. Gelatin zymography was used to assess the activity of the leukocyte MMPs proteins. The obese children showed the following: a) increased expression of leukocyte TIMP-1 and slight elevation (close to statistical significance) of leukocyte MMP-9 (p = 0.054), the decline in MMP-2, b) elevation of plasma MMP-9, leptin, and MMP9/TIMP1 ratio, c) reduced expression of plasma TNF-alpha and MMP-2/TIMP-2 ratio. Several negative correlations were found: TIMP2 vs. ALT (r = -0.536), AST (r = -0.645) and TTG (r = -0.438), IL-6 vs. GGTP (r = -0.815), and MMP12 vs. TTG (r = -0.488), leptin vs. ALT (r = -0.569), MMP-9 vs. total cholesterol (r = -0.556). The only positive correlation was that of plasma leptin level vs. GGTP (r = 0.964). At the beginning of obesity development (children), possibly compensatory reactions prevail, reflected here by an increase in the expression of leukocyte MMPs inhibitor TIMP-1, decrease in the level of leukocyte MMP-2 and plasma MMP-2, MMP2/TIMP-2 ratio, low plasma TNF-alpha and negative correlations between the expression of TIMP-2 and liver (AST, ALT) or fat (TTG) inflammatory markers.

Macrophages coordinate immune response to laser-induced injury via extracellular traps.

Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury. Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina. Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer. Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration.

In-depth human immune cellular profiling from newborn to frail.

Immune functional decline and remodeling accompany aging and frailty. It is still largely unknown how changes in the immune cellular composition differentiate healthy individuals from those become frail at a relatively early age. Our aim in this exploratory study was to investigate immunological changes from newborn to frailty, and the association between health statute and various immune cell subtypes. The participants analyzed in this study covered human cord blood cells and peripheral blood cells collected from young adults, healthy and frail old individuals. A total of 30 immune cell subsets was performed by flow cytometry based on the surface markers of immune cells. Furthermore, frailty was investigated for its relations with various leukocyte subpopulations. Frail individuals exhibited a higher CD4/CD8 ratio, a higher proportion of CD4+ central memory T (TCM) cells, CD8+ effector memory T cells, CD27- switched memory B (CD27-BSM) cells, CD27+ switched memory B cells, age-associated B cells (ABCs) and CD38-CD24- B cells, and a lower proportion of naïve CD8 + T cells and progenitor B cells. The Frailty index score was found to be associated with naïve T cells, CD4/CD8 ratio, ABCs, CD27-BSM cells, and CD4+ TCM cells. Our findings conducted a relatively comprehensive and extensive atlas of age- and frailty-related changes in peripheral leukocyte subpopulations from newborn to frailty. The immune phenotypes identified in this study can contribute to a deeper understanding of immunosenescence in frailty and may provide a rationale for future interventions and diagnosis.

Circulating Leukocyte Subsets Before and After a Breast Cancer Diagnosis and Therapy

Changes in leukocyte composition often precede chronic disease onset. Patients with a history of breast cancer (hereinafter referred to as breast cancer survivors) are at increased risk for subsequent chronic diseases, but the long-term changes in peripheral leukocyte composition following a breast cancer diagnosis and treatment remain unknown. To examine longitudinal changes in peripheral leukocyte composition in women who did and did not develop breast cancer and identify whether differences in breast cancer survivors were associated with specific treatments. In this prospective cohort study, paired blood samples were collected from 2315 women enrolled in The Sister Study, a US-nationwide prospective cohort study of 50 884 women, at baseline (July 2003 to March 2009) and follow-up (October 2013 to March 2015) home visits, with a mean (SD) follow-up interval of 7.6 (1.4) years. By design, approximately half of the included women had been diagnosed and treated for breast cancer after enrollment and before the second blood draw. A total of 410 women were included in the present study, including 185 breast cancer survivors and 225 who remained free of breast cancer over a comparable follow-up period. Data were analyzed from April 21 to September 9, 2022. Breast cancer status and, among breast cancer survivors, cancer treatment type (chemotherapy, radiotherapy, endocrine therapy, or surgery). Blood DNA methylation data were generated in 2019 using a genome-wide methylation screening tool and deconvolved to estimate percentages of 12 circulating leukocyte subsets. Of the 410 women included in the analysis, the mean (SD) age at enrollment was 56 (9) years. Compared with breast cancer-free women, breast cancer survivors had decreased percentages of circulating eosinophils (-0.45% [95% CI, -0.87% to -0.03%]; P = .03), total CD4+ helper T cells (-1.50% [95% CI, -2.56% to -0.44%]; P = .01), and memory B cells (-0.22% [95% CI, -0.34% to -0.09%]; P = .001) and increased percentages of circulating naive B cells (0.46% [95% CI, 0.17%-0.75%]; P = .002). In breast cancer survivor-only analyses, radiotherapy was associated with decreases in total CD4+ T cell levels, whereas chemotherapy was associated with increases in naive B cell levels. Surgery and endocrine therapy were not meaningfully associated with leukocyte changes. In this cohort study of 410 women, breast cancer survivors experienced lasting changes in peripheral leukocyte composition compared with women who remained free of breast cancer. These changes may be related to treatment with chemotherapy or radiotherapy and could influence future chronic disease risk.

A271 EFFECT OF ANTI-TNF AGENTS ON DNA METHYLATION IN PERIPHERAL BLOOD OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE

Abstract Background Despite the success and ongoing use of tumor necrosis factor inhibitors (TNFi) in the treatment of inflammatory bowel disease (IBD), there are no predictors of response to therapy. While much research has gone into understanding genetic risks for IBD, less work has been done exploring epigenetic associations with treatment. Hence, we wondered whether epigenetic changes were associated with failure of TNFi in IBD. Aims Describe the association of TNFi response with DNA methylation in patients with IBD. Methods Participants ≥18 years with IBD (N=169) were selected from the Inflammation, Microbiome, and Alimentation: Gastro-Intestinal and Neuropsychiatric Effects (IMAGINE) Strategy for Patient Oriented Research (SPOR) Network. At enrollment participants completed a questionnaire that included their disease diagnosis, therapy, smoking history, and disease activity using the validated IBD Symptom Inventory (IBDSI). Patients provided whole blood samples that were processed for and run on Illumina DNA methylation arrays. Results The 169 patients were in three treatment groups: TNFi naïve (N=98, never exposed to an anti-TNF agent), TNFi responder (N=32, inactive disease on anti-TNF agent), and TNFi nonresponder (N=39, active disease not on any biologic at time of enrollment). The mean symptom score (SIBDSI) was different across the three groups (pampersand:003C0.001): TNFi nonresponders having a higher SIBDSI (24 ± 6.1) than the TNFi naïve (9.1 ± 5.5) or responder (8.3 ± 4.3) treatment groups. Relative proportions of leukocyte populations were estimated using DNA methylation. CD4 and CD8 T cell and B cell counts were higher (pampersand:003C0.05) in the nonresponders and responders versus TNFi naïve group. Neutrophil counts were lower (pampersand:003C0.05) in the TNFi nonresponders and responders compared to the TNFi naïve group (Figure 1). There was a trend towards increased epigenetic estimates of age acceleration (pampersand:003E0.05) in nonresponders versus responders and TNFi naïve patients, likely driven by disease activity (data not shown). Epigenome-wide analysis of the three groups revealed 16 CpGs for responder versus naïve (e.g. CDK5 regulatory subunit-associated protein 1-like 1 (CDKAL1), and two CpGs for nonresponders versus responders (Table 1). This includes a CpG for guanine nucleotide-binding protein subunit gamma-2 (GNG2) that was shared between the two comparisons. Conclusions Exposure to TNFi is associated with changes in peripheral leukocyte populations independent of response to treatment, indicating a TNFi effect. This implies that anti-TNF treatment is having some effect on patients even if there is clinical nonresponse. Differentially expressed CpGs implicates possible markers of response to treatment, particularly CDKAL1, a gene that has been associated with TNFi response in psoriasis. Funding Agencies Guts and Roses Charity

A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease

IntroductionA novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). Materials and methodsVWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. ResultsThe mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. ConclusionsThe VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

Features of baseline and lipopolysaccharide-induced cytokine secretion in mononuclear leukocyte cultures from patients with the erythema migrans form of acute lyme borreliosis based on clinical parameters

Introduction. Features of cytokine production in mononuclear cell cultures from Lyme borreliosis patients based on clinical data remained poorly studied. The study aim was to estimate the patterns of baseline and lipopolysaccharide-induced cytokine-secretory activity of peripheral blood mononuclear leukocytes from patients with erythema migrans form of acute Lyme borreliosis based on clinical parameters. Materials and methods. Groups of 22 and 12 patients with the diagnoses of mild or moderate severity of monoinfection and co-infection with tick-borne encephalitis of Lyme borreliosis with erythema migrans were examined twice: on week 1 after disease onset and day 14. The control group included 17 healthy donors. Basal and lipopolysaccharide-induced IL-6, IL-10, and TNFα secretion levels were assessed in mononuclear leukocyte culture supernatants applying enzyme immunoassay. Statistical analysis was performed by using the Mann–Whitney U-test, Wilcoxon test, and Spearman’s rank correlation. Results. The group of moderate severity patients was clinically distinguished by severer fever and intoxication manifestations. At the disease onset, the basal TNFα, IL-6 and IL-10 secretion levels in the moderate severity patient cultures were significantly higher than in those of the other groups. After antibiotics treatment, the baseline TNFα and IL-10 levels tended to decrease. At the onset, lipopolysaccharide-induced cultures from the moderate severity patients showed significantly suppressed TNFα production and increased IL-10 secretion as compared to the other groups. Lipopolysaccharide-induced IL-6 secretion in the moderate vs. mild severity group supernatants was significantly lower. In dynamics, the induced TNFα levels in the moderate severity patients were increased to the magnitude exceeding that in the controls. Positive correlations between the IL-6 and TNFα basal levels and maximum body temperature or the C-reactive protein serum concentrations were revealed in the patients. Induced TNFα levels showed negative correlations with fever levels or with IL-10 secretion. Conclusions. It was demonstrated that basal TNFα, IL-6 and IL-10 secretion levels in the mononuclear cell cultures of acute Lyme borreliosis patients increased with the increasing disease severity. Suppression of lipopolysaccharide-induced TNFα production in the moderate severity patient cultures was presumably associated with the regulatory cytokine IL-10 effects.