Response Of Peripheral Blood Leukocytes Research Articles

Single-cell RNA sequencing reveals peripheral blood leukocyte responses to spinal cord injury in mice with humanised immune systems

Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were ‘SCI-specific’ (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.

Development, characterization, and immunomodulation performance of spray-dried Moringa oleifera seed extract in Longfin yellowtail Seriola rivoliana.

Moringa oleifera is one of the most promising plants in aquaculture because it improves the health status, zootechnical parameters and resistance against diseases. This research evaluates the physicochemical, antioxidant values of spray-dried Moringa oleifera seed extract microencapsulates obtained at 140 and 180°C with whey protein concentrate (WPC) and maltodextrin (MD) as wall materials in two different proportions: WPC 100% and WPC-MD (3:1). Also, immune response of peripheral blood leukocytes (PBL) of Longfin yellowtail Seriola rivoliana stimulated with spray-dried Moringa oleifera seed for 24h was assessed. The physicochemical parameters show that the recovery yield for all the treatments was of 65% and microencapsulates demonstrated to be stable in the physicochemical tests with low solubilization times and protection against humidity. For WPC-MD (3:1)/140°C, bioactive compound retention and antioxidant potential were higher than in other combinations. The immunological test show that any treatments was non-cytotoxic against peripheral blood leukocytes. WPC-MD (3:1)/140°C treatment enhanced immune parameters as phagocytosis, respiratory burst, myeloperoxidase activities and nitric oxide production. Immune related genes as IL-1β and TNF-α were up-regulated in those stimulated leukocytes with WPC-MD (3:1)/140°C. The results suggest that this combination may be a good alternative for animal health as a medicinal and immunostimulant additive.

Japanese Flounder HMGB1: A DAMP Molecule That Promotes Antimicrobial Immunity by Interacting with Immune Cells and Bacterial Pathogen.

High mobility group box (HMGB) proteins are DNA-associated proteins that bind and modulate chromosome structures. In mammals, HMGB proteins can be released from the cell nucleus and serve as a damage-associated molecular pattern (DAMP) under stress conditions. In fish, the DAMP function of HMGB proteins in association with bacterial infection remains to be investigated. In this study, we examined the immunological functions of two HMGB members, HMGB1 and HMG20A, of Japanese flounder. HMGB1 and HMG20A were expressed in multiple tissues of the flounder. HMGB1 was released from peripheral blood leukocytes (PBLs) upon bacterial challenge in a temporal manner similar to that of lactate dehydrogenase release. Recombinant HMGB1 bound to PBLs and induced ROS production and the expression of inflammatory genes. HMGB1 as well as HMG20A also bound to various bacterial pathogens and caused bacterial agglutination. The bacteria-binding patterns of HMGB1 and HMG20A were similar, and the binding of HMGB1 competed with the binding of HMG20A but not vice versa. During bacterial infection, HMGB1 enhanced the immune response of PBLs and repressed bacterial invasion. Collectively, our results indicate that flounder HMGB1 plays an important role in antimicrobial immunity by acting both as a modulator of immune cells and as a pathogen-interacting DAMP.

Sex Differences in Innate Immune Response of Peripheral Blood Leukocytes of Alzheimer's Disease Patients.

Neurodegenerative disorders, including Alzheimer's disease (AD), are associated with a disruption of normal immune function that could potentially impact the brain. In AD sex and gender have been noted as relevant to disease prevalence or clinical manifestation. It is suggested that disease progression could vary as a result of the different inflammation state among males and females. The objective was to investigate sex-dependent difference in innate immunity of AD patients and healthy, age-matched controls. The level of innate immunity was measured with test based on peripheral blood leukocytes (PBLs) resistance to viral infection (vesicular stomatitis virus, VSV) ex vivo. Cytokine: TNF-α, IFN-γ, IL-1β, IL-10 production by uninfected and VSV-infected PBLs ex vivo with enzyme-linked immunosorbent assay were examined. In contrast to controls, women with AD exhibit lower average level of innate immunity than AD men. The mean level of TNF-α, IL-10 and IL-1β was higher in AD men than in AD women whereas such changes were not observed among controls. The level of IFN-γ was higher in AD than in controls. PBLs from AD did not increase IFN-γ production after viral infection in contrast to controls. Leukocytes from women with AD exhibited a weaker response to viral infection and much less cytokine production compared to men with AD. It is important to consider sex as a biological variable in AD as it shows promises to advance our understanding of mechanisms of AD pathology and may be the basis for future treatment of AD.

Ginkgo Biloba Leaf Extract Improves an Innate Immune Response of Peripheral Blood Leukocytes of Alzheimer’s Disease Patients

Background: One of the main features of Alzheimer’s disease (AD) pathology is failure in innate immune response and chronic inflammation. Lack of effective AD treatment means that more attention is paid to alternative therapy and drugs of natural origin, such as extract of Ginkgo biloba (EGb). The purpose of this study was to investigate the effect of EGb on the mechanisms of innate immune response of peripheral blood leukocytes (PBLs) in AD patients. Methods: In AD patients and healthy-age matched controls, the effect of EGb on two of innate immune reactions, i.e., PBLs resistance to viral infection ex vivo and production of cytokines, namely TNF-α, IFN-γ, IL-1β, IL-10, IL-15, and IFN-α, were investigated. The influence of EGb on inflammatory-associated genes expression that regulate innate immune response to viral infection and cytokine production, namely IRF-3, IRF-7, tetherin, SOCS1, SOCS3, NFKB1, p65, and MxA was also examined. Results: A beneficial effect of EGb especially in AD women was observed. EGb decreased production of TNF-α, IFN-γ, and IL-10 and increased IL-15 and IL-1β. The effect was more pronouncement in AD group. EGb also downregulated expression of investigated genes. Conclusions: EGb may have an advantageous properties for health management in elderly and AD sufferers but especially in women with AD. Improving peripheral innate immune cells’ activity by adding EGb as accompanying treatment in AD may be, in the long term, a good course to modify the disease progression.

Dietary supplementation of Bacillus velezensis improves Vibrio anguillarum clearance in European sea bass by activating essential innate immune mechanisms

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

Sex-specific differences in peripheral blood leukocyte transcriptional response to LPS are enriched for HLA region and X chromosome genes

Sex-specific differences in prevalence are well documented for many common, complex diseases, especially for immune-mediated diseases, yet the precise mechanisms through which factors associated with biological sex exert their effects throughout life are not well understood. We interrogated sex-specific transcriptional responses of peripheral blood leukocytes (PBLs) to innate immune stimulation by lipopolysaccharide (LPS) in 46 male and 66 female members of the Hutterite community, who practice a communal lifestyle. We identified 1217 autosomal and 54 X-linked genes with sex-specific responses to LPS, as well as 71 autosomal and one X-linked sex-specific expression quantitative trait loci (eQTLs). Despite a similar proportion of the 15 HLA genes responding to LPS compared to all expressed autosomal genes, there was a significant over-representation of genes with sex by treatment interactions among HLA genes. We also observed an enrichment of sex-specific differentially expressed genes in response to LPS for X-linked genes compared to autosomal genes, suggesting that HLA and X-linked genes may disproportionately contribute to sex disparities in risk for immune-mediated diseases.

The Mechanisms of Sijunzi Decoction in the Treatment of Chronic Gastritis Revealed by Network Pharmacology.

Chronic gastritis is characterized by inflammation in the gastric mucosa with a vicious circle in inflammatory cells and inflammatory mediators. Stomach adenocarcinoma would occur in the metaplastic gastric mucosa of chronic gastritis. Sijunzi decoction is a famous classical formula for the treatment of chronic gastritis. Although previous studies revealed some functions of Sijunzi decoction in treating chronic gastritis, the underlying mechanisms have not been illustrated clearly. In this study, we used network pharmacology to investigate the mechanism of Sijunzi decoction in treating chronic gastritis. Firstly, online datasets TCMSP, SWISS, and DisGeNET were used to investigate the functional mechanism of Sijunzi decoction against chronic gastritis and 18 genes were identified as targets of Sijunzi decoction in chronic gastritis. These 18 genes can be categorized into immunologically related genes and cancer-related genes. GO analysis showed that the 18 target genes were mainly enriched in angiogenesis, nitric oxide biosynthetic process, ERK1 and ERK2 cascade, cellular response to drug, and MAPK cascade. So, Sijunzi decoction alleviated chronic gastritis by inhibiting the local inflammatory response. Furthermore, we also investigated the impact of Sijunzi decoction on the peripheral blood leukocytes with our own RNA sequencing (RNA-seq) data of 11 chronic superficial gastritis patients. 102 differentially expressed genes (DEGs) were identified by comparing RNA-seq data of chronic superficial gastritis patients with healthy control groups. After performing a functional analysis on 102 DEGs and Sijunzi decoction potential targets and taking the intersection of these pathways, we found that platelet activation, angiogenesis, and pathways in cancer were candidate target pathways regulated by Sijunzi decoction. Thus, Sijunzi decoction also alleviates chronic gastritis by suppressing inflammatory response of peripheral blood leukocytes. Our results showed that Sijunzi decoction can ameliorate the local gastric inflammation and inflammations in peripheral blood leukocytes and might also reduce the incidence of stomach cancer in chronic gastritis.

Identification of novel molecular markers of mastitis caused by Staphylococcus aureus using gene expression profiling in two consecutive generations of Chinese Holstein dairy cattle

BackgroundMastitis in dairy cows caused by Staphylococcus aureus is a major problem hindering economic growth in dairy farms worldwide. It is difficult to prevent or eliminate due to its asymptomatic nature and long persistence of infection. Although transcriptomic responses of bovine mammary gland cells to pathogens that cause mastitis have been studied, the common responses of peripheral blood leukocytes to S. aureus infection across two consecutive generations of dairy cattle have not been investigated.MethodsIn the current study, RNA-Seq was used to profile the transcriptomes of peripheral blood leukocytes sampled from S. aureus-infected mothers and their S. aureus-infected daughters, and also healthy non-infected mothers and their healthy daughters. Differential gene expression was evaluated as follows: 1) S. aureus-infected cows versus healthy non-infected cows (S vs. H, which include all the mothers and daughters), 2) S. aureus-infected mothers versus healthy non-infected mothers (SM vs. HM), and 3) S. aureus-infected daughters versus healthy non-infected daughters (SMD vs. HMD).ResultsAnalysis of all identified expressed genes in the four groups (SM, SMD, HM, and HMD) showed that EPOR, IL9, IFNL3, CCL26, IL26 were exclusively expressed in both the HM and HMD groups, and that they were significantly (P < 0.05) enriched for the cytokine-cytokine receptor interaction pathway. A total of 17, 13 and 10 differentially expressed genes (DEGs) (FDR Padj. < 0.1 and |FC| > 1.2) were detected in the three comparisons, respectively. DEGs with P < 0.05 and |FC| > 2 were used for functional enrichment analyses. For the S vs. H comparison, DEGs detected included CCL20, IL13 and MMP3, which are associated with the IL-17 signaling pathway. In the SM vs. HM and SMD vs. HMD comparisons, five (BLA-DQB, C1R, C2, FCGR1A, and KRT10) and six (BLA-DQB, C3AR1, CFI, FCAR, FCGR3A, and LOC10498484) genes, respectively, were involved in the S. aureus infection pathway.ConclusionsOur study provides insights into the transcriptomic responses of bovine peripheral blood leukocytes across two generations of cattle naturally infected with S. aureus. The genes highlighted in this study could serve as expression biomarkers for mastitis and may also contain sequence variation that can be used for genetic improvement of dairy cattle for resilience to mastitis.

Changes in peripheral inflammation-related gene expression by postprandial glycemic response in healthy Japanese men

ObjectivesContinuous postprandial hyperglycemia is associated with the onset of cardiovascular disease. In recent years, the mRNA expression of inflammation-related genes in peripheral blood leukocytes has been shown to be induced by an increase in blood glucose levels. The aim of this study was to investigate differences in the expression of inflammation-related genes in peripheral blood leukocytes in response to an increase in blood glucose from individuals who consumed two kinds of breakfast meals with different glycemic indexes (GIs). MethodsTwenty healthy Japanese men 40 to 70 y of age were given low- or high-GI meals for breakfast for 14 d. Clinical examinations were performed on days 7 and 14. Their blood glucose levels and insulin concentrations were measured from before breakfast ingestion to 120 min after. Additionally, using the blood obtained before and 120 min after breakfast, the mRNA expression levels of inflammation-related genes in peripheral leukocytes were measured. ResultsThe blood glucose levels were significantly lower in the low-GI meal intake group at 30, 60, and 120 min after breakfast than in the high-GI meal intake group. The intake of high-GI meals for 6 d led to an increase in the mRNA levels of interleukin-1β, S100A4, and CD18 compared with the period of low-GI meals. ConclusionThe intake of a low-GI breakfast for 1 wk in healthy Japanese men resulted in lower postprandial blood glucose and insulin levels, which were accompanied by a reduced expression of inflammation-related genes in peripheral blood leukocytes.

Analysis of the Leukocyte Response in Calves Suffered from Mycoplasma bovis Pneumonia.

Mycoplasma bovis is known to be a cause of chronic pneumonia in cattle. To date, the disease pathomechanism has not been fully elucidated. Leukocytes play a key role in host antimicrobial defense mechanisms. Many in vitro studies of the effect of Mycoplasma bovis (M. bovis) on leukocytes have been performed, but it is difficult to apply these results to in vivo conditions. Additionally, only a few studies on a local immune response in M. bovis pneumonia have been undertaken. In this study, the experimental calf-infection model was used to determine the effect of field M. bovis strains on changes of the peripheral blood leukocyte response, including phagocytic activity and oxygen metabolism by cytometry analyses. An additional aim was to evaluate the lung local immunity of the experimentally infected calves using immunohistochemical staining. The general stimulation of phagocytic and killing activity of peripheral blood leukocytes in response to the M. bovis infection points to upregulation of cellular antimicrobial mechanisms. The local immune response in the infected lungs was characterized by the T- and B-cell stimulation, however, most seen in the increased T lymphocyte response. Post-infection, strong expression of the antigen-presenting cells and phagocytes also confirmed the activation of lung local immunity. In this study—despite the stimulation—both the peripheral and local cellular antimicrobial mechanisms seem to appear ineffective in eliminating M. bovis from the host and preventing the specific lung lesions, indicating an ability of the pathogen to avoid the host immune response in the M. bovis pneumonia.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.

TLR4 and TLR2 activation is differentially associated with age during Parkinson’s disease

ABSTRACTParkinson’s disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons associated with neuroinflammation. Toll-like receptors (TLRs) are expressed in peripheral blood leukocytes and also in neurons and glial cells mediating inflammation. This study aimed to investigate the peripheral blood leukocyte response to TLR2 and TLR4 agonists in young and elderly PD patients. Two groups of patients with PD were evaluated (≤ 55 years old and ≥ 65 years old), age-matched with healthy controls (n = 26). Severity of PD was evaluated by Unified Parkinson’s Disease Rating Scale (UPDRS). Whole blood cultures were stimulated with lipopolysaccharide (LPS), a TLR4 agonist or Pam3Cys (Pam), a TLR2 agonist. Tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) were measured by immunoenzimatic assay. 6 h-TNFα production was increased after TLR4 stimulation, mainly in young PD patients, whereas TLR2-induced TNFα and IL-10 levels were decreased in PD patients independent of age (p < 0.05). A reverse correlation between LPS-induced TNFα production and age was observed in PD patients and controls, but TNFα induced by TLR2 agonist was not associated with age of PD patients or controls. TNFα production induced by TLR4 but not by TLR2 was reversely associated with the age at PD onset and disease duration. No associations between UPDRS scores and cytokine levels were detected. In conclusion, TLR4 and TLR2 responses seem to be differentially affected during PD. Data suggest that TLR2 deficiency in periphery is independent of age of the patients, age at PD onset, or PD duration.

The common promoter polymorphism rs11666254 downregulates FPR2/ALX expression and increases risk of sepsis in patients with severe trauma

BackgroundFormyl peptide receptor 2-lipoxin receptor (FPR2/ALX) modulates the anti-inflammatory response and therefore may be a target for treating sepsis. The purpose of this study was to investigate the association between genetic variants of the FPR2/ALX gene and sepsis after severe trauma as well as to further analyze the functions of sepsis-related genetic polymorphisms.MethodsThree tag single-nucleotide polymorphisms (tag SNPs) that captured all common alleles across the FPR2/ALX genomic region were genotyped using pyrosequencing in an initial sample consisting of 275 patients with severe trauma. The rs11666254 polymorphism, which had statistical significance, was genotyped in an additional 371 patients, and logistic regression analysis was performed to determine associations between the FPR2/ALX gene polymorphism and sepsis susceptibility after severe trauma. The messenger RNA (mRNA) and protein levels of FPR2/ALX in the lipopolysaccharide-stimulated white blood cells of trauma patients were determined by performing quantitative polymerase chain reactions and Western blot analysis. Tumor necrosis factor (TNF)-α production was measured by enzyme-linked immunosorbent assay. The effects of the promoter polymorphism rs11666254 on the transcription activity of FPR2/ALX were analyzed using a luciferase reporter assay.ResultsAmong the three tag SNPs, only the rs11666254 polymorphism was found to be significantly associated with sepsis in trauma patients, and this association persisted after a pooled analysis of all 646 trauma patients, which showed that patients who carried the A allele of rs11666254 had a significantly higher risk of developing sepsis than individuals who carried the G allele. This SNP was also significantly associated with lower FPR2/ALX mRNA and protein expression as well as higher TNF-α production from the peripheral blood leukocyte response to bacterial lipoprotein stimulation. In addition, the rs11666254 polymorphism could significantly decrease the promoter activity of the FPR2/ALX gene.ConclusionsThe rs11666254 polymorphism in the FPR2/ALX gene is a functional SNP that increases sepsis susceptibility in patients after traumatic injury.

Pregnant human peripheral leukocyte migration during several late pregnancy clinical conditions: a cross-sectional observational study

BackgroundParturition at term and preterm is characterized by sterile inflammatory processes occurring in the absence of infection whereby peripheral leukocytes infiltrate gestational tissues in response to chemotactic signals. In response to a homing signal, recruited leukocytes undergo diapedesis and extravasate through capillaries, migrating into stromal tissue. There they interact with resident immune and stromal cells to produce a mixture of matrix metalloproteinases, prostaglandins and cytokines including interleukin-1β (IL-1β) and IL-6 that in turn transform the uterus from pregnancy to parturition. Since migration is an early parturitional event our purpose was to study the migration of maternal peripheral blood leukocytes in response to a standard chemotactic signal during several different conditions of late pregnancy.MethodsWe used a cross-sectional observational study design. Subjects were (sTL) spontaneous normal labour delivered vaginally at term, (TNL) elective caesarean section at term without labour, (PTL) preterm in labour, (PTNL) preterm not in labour, (TPTL) threatened preterm labour, and (pPROM) preterm with premature rupture of membranes. Leukocytes (100,000) obtained by venipuncture and chemotactic factor isolated from term labour fetal membranes were placed in the upper and lower halves, respectively, of a Boyden chamber separated by a filter with 3μm pores. Migrated leukocytes were assessed by flow cytometry. The number of leukocytes that migrated in 90 min was the primary outcome measure.ResultsIncreased numbers of leukocytes from peripheral blood of women in labour (TL or PTL) or soon to go into labour (PPROM) migrated towards a chemotactic signal than did leukocytes from women not in labour (TNL, PTNL, or TPTL) (p < 0.0001). All pPROM delivered within 7d; TPTL delivered >30d. Receiver operating characteristic curve parameters indicated the cut-off point for delivery within 7d to be 37,082 leukocytes with sensitivity 78.1%, specificity 88.9%, positive predictive value 91.4%, negative predictive value 72.7%, and area under the curve 0.83.ConclusionLeukocyte migration to a fetal membrane signal varies in a predictable fashion during various clinical situations of late gestation. This principle has the potential to be improved to become a clinical test to predict delivery.

Abstract # 1805 Peripheral cutaneous inflammation induces a brain chemokine response, immune cell infiltration and functional changes

There are now compelling data linking peripheral inflammation and depression. However, the mechanisms that underlie this relationship remain unclear. Using QPCR, we measured brain, lung, liver, skin and peripheral blood leukocyte responses to a well-characterised murine model of psoriasis-like skin inflammation, Aldara (containing the TLR-7 ligand, Imiquimod), in C57BL/6 female mice. We measured tissue responses across a number of time points (4 h, 12 h, 1 d, 3 d and 5 d) and confirmed qPCR findings with protein analysis of brain tissue. Further, we measured behavioural outcomes using burrowing, a rewarding behaviour, deficits in which can be construed as anhedonic. We found significant transcriptional upregulation of a number of chemokines and pro-inflammatory cytokines (including CCL2, CCL5, CXCL10, IL-1B, IL-6 and TNF-a) in the brain and systemic organs and concurrent upregulation of protein was confirmed in the brain. However, the brain response was temporally unique in that the peak transcriptional response was observed at 3 days compared to 12 h to 1 day in the peripheral tissues. Along with chemokine and cytokine induction, we observed the infiltration of immune cells into the brain, including T cells, NK cells and monocytes, particularly at day 3 and day 5. These data indicate that the brain mounts a unique response to peripheral inflammatory stimuli, including chemokine induction that may play a role in immune cell recruitment and subsequent behavioural deficits.

Identification of haplotype tag single nucleotide polymorphisms within the nuclear factor-κB family genes and their clinical relevance in patients with major trauma.

IntroductionNuclear factor-κB (NF-κB) family plays an important role in the development of sepsis in critically ill patients. Although several single nucleotide polymorphisms (SNPs) have been identified in the NF-κB family genes, only a few SNPs have been studied.MethodsA total of 753 patients with major blunt trauma were included in this study. Tag SNPs (tSNPs) were selected from the NF-κB family genes (NFKB1, NFKB2, RELA, RELB and REL) through construction of haplotype blocks. The SNPs selected from genes within the canonical NF-κB pathway (including NFKB1, RELA and REL), which played a critical role in innate immune responses were genotyped using pyrosequencing method and analyzed in relation to the risk of development of sepsis and multiple organ dysfunction (MOD) syndrome. Moreover, the rs842647 polymorphism was analyzed in relation to tumor necrosis factor α (TNF-α) production by peripheral blood leukocytes in response to bacterial lipoprotein stimulation.ResultsEight SNPs (rs28362491, rs3774932, rs4648068, rs7119750, rs4803789, rs12609547, rs1560725 and rs842647) were selected from the NF-κB family genes. All of them were shown to be high-frequency SNPs in this study cohort. Four SNPs (rs28362491, rs4648068, rs7119750 and rs842647) within the canonical NF-κB pathway were genotyped, and rs842647 was associated with sepsis morbidity rate and MOD scores. An association was also observed between the rs842647 A allele and lower TNF-α production.Conclusionsrs842647 polymorphism might be used as relevant risk estimate for the development of sepsis and MOD syndrome in patients with major trauma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0836-6) contains supplementary material, which is available to authorized users.

Activation of the TLR1/2 pathway induces the shaping of the immune response status of peripheral blood leukocytes.

Toll-like receptors (TLRs) play an essential role in the activation and regulation of the innate and adaptive immune responses through the recognition of specific components of pathogens. TLR1/2 on the cell surface plays an important role in defending against Gram-positive bacteria. The aim of the present study was to examine the expressional variation of immunomodulatory molecules in peripheral blood leukocytes (PBLs) treated with the TLR1/2 agonist, Pam3Cys. The quantitative polymerase chain reaction result showed dramatically increased expression of immune-related factors treated with Pam3Cys. Antibody-chip assays confirmed that activation of TLR1/2 could induce secretion of four important immune factors [interleukin (IL)-6, IL-8, macrophage inflammatory protein-1α and interferon-β). Western-blot analysis indicated the upregulation of three significant signal kinase proteins (phosphorylated signal transducer and activator of transcription 3, extracellular signal-related kinase and c-Jun N-terminal kinase 2). The study demonstrated that there were numerous molecules involved in the immune response of PBLs stimulated by the TLR1/2 ligand. Our future studies will focus on the mechanisms of these molecules in the TLR1/2 agonist-mediated immune response.

Contrasting the Microbiomes From Healthy Volunteers and Patients With Chronic Rhinosinusitis

Chronic rhinosinusitis (CRS) is the persistent inflammation of the sinus and nasal passages lasting over 3 months. The etiology of CRS is not well understood. To obtain insights into the disease process, we contrasted the microbiome and immune response from patients with CRS and healthy controls. A case vs control design was used. Samples were collected in the operating room in an institutional hospital or clinic. Thirty patients with CRS and 12 healthy controls undergoing surgery were recruited. The microbiome was analyzed by deep sequencing of the bacterial 16S and fungal 18S ribosomal RNA genes. Immune response was measured by quantification of 30 different cytokines by multiplexed enzyme-linked immunosorbent assay, and immune cells in the lavage were identified by flow cytometry. The immune response of peripheral blood leukocytes to the lavage microbiota was assessed by interleukin (IL)-5 enzyme-linked immunospot assay. While quantitative increase in most bacterial and fungal species was observed in patients with CRS relative to controls, the microbiomes of patients with CRS were qualitatively similar to the controls. Because these results indicated that bacteria and fungi are not triggering CRS, we undertook a more detailed characterization of the immune response. Patients with CRS had increased levels of the following cytokines: IL-4, IL-5, IL-8, and IL-13, along with increased levels of eosinophils and basophils in the lavage. Importantly, peripheral blood leukocytes isolated from patients with CRS responded to control lavage samples (ie, to commensals) to produce IL-5. In contrast, the same lavage sample evoked no IL-5 production in leukocytes from healthy controls. These findings support the theory that in some cases CRS results from an immune hyperresponsiveness to commensal organisms.

First characterization of a teleost Epstein-Barr virus-induced gene 3 (EBI3) reveals a regulatory effect of EBI3 on the innate immune response of peripheral blood leukocytes

Epstein-Barr virus-induced gene 3 (EBI3) encodes a protein that in mammals is known to be a subunit of interleukin (IL)-27 and IL-35, both which regulate cytokine production and inflammatory response. To date, no studies on fish EBI3 have been documented. In this work, we report the identification of an EBI3 homologue, CsEBI3, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsEBI3 is composed of 245 amino acid residues and possesses a Fibronectin type 3 (FN3) domain that is preserved in lower and higher vertebrates. Expression of CsEBI3 was detected in a wide range of tissues, in particular those of immune relevant organs, and upregulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) enhanced CsEBI3 expression and caused extracellular secretion of CsEBI3. Purified recombinant CsEBI3 (rCsEBI3) stimulated the respiratory burst activity of PBL and upregulated the expression of IL-1β, IL-8, Myd88, interferon-induced gene 15, CD28, and chemokines. In contrast, rCsEBI3M, a mutant CsEBI3 that lacks the FN3 domain failed to activate PBL and induced much weaker expression of the immune genes. Treatment of PBL with rCsEBI3, but not with the mutant rCsEBI3M, enhanced cellular resistance against bacterial invasion, whereas antibody blocking of CsEBI3 on PBL significantly reduced cellular resistance against bacterial infection. Taken together, these results indicate for the first time that a teleost EBI3 possesses immunoregulatory property in a manner that is dependent on the conserved FN3 domain, and that CsEBI3 is involved in the innate immune defense of PBL against microbial pathogens.