Number Of Circulating Leukocytes Research Articles

GLP-2 predicts cardiovascular outcomes in patients with myocardial infarction and increases atherosclerosis in mice

Abstract Background GLP-1 and GLP-2 (glucagon-like peptide-1/2) are incretin hormones that are co-secreted from intestinal L-cells in response to food intake and inflammatory stimuli. While GLP-1 is known to induce postprandial insulin secretion and to improve cardiovascular outcomes in patients with diabetes, GLP-2 enhances intestinal nutrient absorption. GLP-2 agonists are clinically used for the treatment of patients with short bowel syndrome. The relevance of GLP-2 beyond the gut is not well understood. The aim of this study was to investigate the role of GLP-2 for cardiovascular disease (CVD). Methods Total GLP-2 levels were assessed at time of admission in 918 patients with myocardial infarction presenting with acute chest pain, among them 597 patients with NSTEMI and 321 with STEMI. The primary composite outcome of the study was the first occurrence of all-cause death, nonfatal myocardial infarction, or nonfatal stroke (3-P-MACE) with a median follow-up of 311 days. To induce atherosclerosis, Glp2r−/− or WT mice were injected with PCSK9 virus and fed a diet high in cholesterol (HCD) for 12 weeks. Results Kaplan-Meier survival plots (separated by the median of GLP-2 with a cut-off value of 4.4 pM) and univariable cox regression analyses found GLP-2 values to be associated with adverse outcome (3-P-MACE and all-cause mortality; logarithmized GLP-2 values HR: 2.87; p<.0001). Further adjustment for age, sex, smoking, hypertension, hypercholesterinemia, previous CVD and diabetes mellitus did not affect this association (logarithmized GLP-2 values HR: 2.66; p=0.0055). Receiver operating characteristic curve (ROC) analyses illustrated that GLP-2 is a strong indicator for early events (area under the curve of the combined endpoint at 7 days: 0.74; 14 days: 0.76; 30 days: 0.76; 6 months: 0.72), which proved to be superior to Troponin T and hs-CRP. To asses the functional role of GLP-2 in CVD in an experimental approach we injected Glp2r−/− or WT mice PCSK9 virus (to induce functional Ldlr-deficiency and hypercholesterolemia) and fed these mice a HCD. After 12 weeks Glp2r−/− mice compared to WT littermates presented with a significant reduction in plaque volume and lesion size. While body weight and circulating leukocyte numbers (FACS analysis) were unaffected, Glp2r−/− mice had lower cholesterol levels. Conclusion Circulating GLP-2 levels are associated with cardiovascular events in patients with acute myocardial infarction while inactivation of the GLP-2 system reduces atherosclerosis in mice. Future studies are needed to investigate whether the GLP-2 receptor might provide a novel therapeutic target for cardiovascular disease.

The role of platelet P2Y12 receptors in inflammation.

Inflammation is a complex pathophysiological process underlying many clinical conditions. Platelets contribute to the thrombo-inflammatory response. Platelet P2Y12 receptors amplify platelet activation, potentiating platelet aggregation, degranulation and shape change. The contents of platelet alpha granules, in particular, act directly on leucocytes, including mediating platelet-leucocyte aggregation and activation via platelet P-selectin. Much evidence for the role of platelet P2Y12 receptors in inflammation comes from studies using antagonists of these receptors, such as the thienopyridines clopidogrel and prasugrel, and the cyclopentyltriazolopyrimidine ticagrelor, in animal and human experimental models. These suggest that antagonism of P2Y12 receptors decreases markers of inflammation with some evidence that this reduces incidence of adverse clinical sequelae during inflammatory conditions. Interpretation is complicated by pleiotropic effects such as those of the thienopyridines on circulating leucocyte numbers and of ticagrelor on adenosine reuptake. The available evidence suggests that P2Y12 receptors are prominent mediators of inflammation and P2Y12 receptor antagonism as a potentially powerful strategy in a broad range of inflammatory conditions. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Rivaroxaban attenuates neutrophil maturation in the bone marrow niche

Pharmacological inhibition of factor Xa by rivaroxaban has been shown to mediate cardioprotection and is frequently used in patients with, e.g., atrial fibrillation. Rivaroxaban's anti-inflammatory actions are well known, but the underlying mechanisms are still incompletely understood. To date, no study has focused on the effects of rivaroxaban on the bone marrow (BM), despite growing evidence that the BM and its activation are of major importance in the development/progression of cardiovascular disease. Thus, we examined the impact of rivaroxaban on BM composition under homeostatic conditions and in response to a major cardiovascular event. Rivaroxaban treatment of mice for 7 days markedly diminished mature leukocytes in the BM. While apoptosis of BM-derived mature myeloid leukocytes was unaffected, lineage-negative BM cells exhibited a differentiation arrest at the level of granulocyte–monocyte progenitors, specifically affecting neutrophil maturation via downregulation of the transcription factors Spi1 and Csfr1. To assess whether this persists also in situations of increased leukocyte demand, mice were subjected to cardiac ischemia/reperfusion injury (I/R): 7 d pretreatment with rivaroxaban led to reduced cardiac inflammation 72 h after I/R and lowered circulating leukocyte numbers. However, BM myelopoiesis showed a rescue of the leukocyte differentiation arrest, indicating that rivaroxaban's inhibitory effects are restricted to homeostatic conditions and are mainly abolished during emergency hematopoiesis. In translation, ST-elevation MI patients treated with rivaroxaban also exhibited reduced circulating leukocyte numbers. In conclusion, we demonstrate that rivaroxaban attenuates neutrophil maturation in the BM, which may offer a therapeutic option to limit overshooting of the immune response after I/R.

118 Murine Expression of Humanized Toll-Like Receptor 4 Augments Local and Systemic Immune Responses Following Oropharyngeal Aspiration of Nickel Oxide Nanoparticles

Abstract It has been demonstrated that murine expression of humanized Toll-like receptor-4 (hTLR-4) confers enhanced immunological responsivity to nickel in the skin; however, it remains unclear if hTLR-4 expression also augments nickel-induced immune responses in the lung. hTLR-4 positive/negative mice of both sexes were exposed to vehicle control (dispersion media) or nickel oxide nanoparticles (NiONP; 48 nm; 2.5, 5, or 20 µg) by oropharyngeal aspiration. Mice were euthanized 1, 7, 14, or 28 days post-exposure. Bronchoalveolar lavage (BAL) was performed, blood was collected, and the lung-associated lymph nodes (LALN) and spleen were harvested for phenotypic analysis. By 7d, total BAL cells, lung LDH, and circulating leukocyte number were all significantly elevated in both female/male hTLR-4 positive mice compared to same-sex hTLR-4 negative mice—effects which normalized by 14d. Dose-responsive increases in LALN size were observed in all NiONP-exposed animals by 14d, but no significant differences were observed between hTLR-4 positive/negative animals. The cellular composition of the LALN and spleen, however, was significantly altered at this timepoint between hTLR-4 positive/negative mice of both sexes. In general, hTLR-4 positive mice exhibited selective increases in proportionality and activation status of CD4+ T-cells and B-cells within both tissues. This alteration remained significant at 28d in female mice only. Collectively, these findings suggest that hTLR-4 expression confers significant alterations in early immune responses to NiONP in the lung and associated lymphoid tissues—effects which may render the hTLR-4 mouse model a more translationally-relevant approach for studying respiratory nickel allergy in vivo.

The Role of Neutrophils in Oncolytic Orf Virus-Mediated Cancer Immunotherapy.

Neutrophils are innate leukocytes with diverse effector functions that allow them to respond to pathogens rapidly. Accumulating evidence has highlighted these cells’ complex roles in the host’s response to viral infections and tumor progression. Oncolytic virotherapy is emerging as a promising treatment modality in the armamentarium of cancer therapeutics. Oncolytic viruses preferentially kill cancer cells and stimulate tumor-associated inflammation, resulting in tumor regression. Assessing the activity of individual effector cell subsets following oncolytic virotherapy is important in identifying their contribution to antitumor immunity. In this study, we investigated the role of neutrophils in oncolytic Orf-virus-mediated immunotherapy in a murine model of pulmonary melanoma metastases. The systemic administration of the Orf virus stimulated a dramatic increase in the number of leukocytes in circulation and within the tumor microenvironment, most of which were neutrophils. Analysis of tumor-burdened lungs shortly after therapy revealed significant numbers of phenotypically immature neutrophils, with the enhanced expression of molecules affiliated with activation, migration, and cytotoxicity. Neutrophils stimulated by Orf virus therapy were directly tumoricidal through tumor necrosis factor-α-mediated effects and were required for optimal antitumor efficacy following Orf virus therapy. Taken together, these data reveal neutrophils as a crucial innate effector to consider when investigating oncolytic virotherapy.

Chemokine CCL9 Is Upregulated Early in Chronic Kidney Disease and Counteracts Kidney Inflammation and Fibrosis.

Inflammation and fibrosis play an important pathophysiological role in chronic kidney disease (CKD), with pro-inflammatory mediators and leukocytes promoting organ damage with subsequent fibrosis. Since chemokines are the main regulators of leukocyte chemotaxis and tissue inflammation, we performed systemic chemokine profiling in early CKD in mice. This revealed (C-C motif) ligands 6 and 9 (CCL6 and CCL9) as the most upregulated chemokines, with significantly higher levels of both chemokines in blood (CCL6: 3–4 fold; CCL9: 3–5 fold) as well as kidney as confirmed by Enzyme-linked Immunosorbent Assay (ELISA) in two additional CKD models. Chemokine treatment in a mouse model of early adenine-induced CKD almost completely abolished the CKD-induced infiltration of macrophages and myeloid cells in the kidney without impact on circulating leukocyte numbers. The other way around, especially CCL9-blockade aggravated monocyte and macrophage accumulation in kidney during CKD development, without impact on the ratio of M1-to-M2 macrophages. In parallel, CCL9-blockade raised serum creatinine and urea levels as readouts of kidney dysfunction. It also exacerbated CKD-induced expression of collagen (3.2-fold) and the pro-inflammatory chemokines CCL2 (1.8-fold) and CCL3 (2.1-fold) in kidney. Altogether, this study reveals for the first time that chemokines CCL6 and CCL9 are upregulated early in experimental CKD, with CCL9-blockade during CKD initiation enhancing kidney inflammation and fibrosis.

Residual effects of short-term whole-body cold-water immersion on the cytokine profile, white blood cell count, and blood markers of stress

Background: One of the most challenging environmental extremes is immersion in cold/icy water, and consequent common assumption is that even a brief exposure to cold can lead to cold-related illnesses. The increase in the concentrations of the stress hormones cortisol, epinephrine (Epi), and norepinephrine (NE) in response to acute cold stress are thought to suppress the release of proinflammatory cytokines. No previous study has explored the residual consequences of whole-body short-term cold-water immersion (CWI; 14 °C for 10 min) on the immune response in healthy non-acclimated young adult men (aged 20–30 years). Materials and methods: In the current study, we tested the hypothesis that short-term acute whole-body CWI would induce high blood levels of cortisol, NE, and Epi, which in turn would increase circulating leukocyte numbers and delay the production of proinflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6). Results: Short-term whole-body CWI produced a stressful physiological reaction, as manifested by hyperventilation and increased muscle shivering, metabolic heat production, and heart rate. CWI also induced the marked release of the stress hormones Epi, NE, and cortisol. The change in IL-6 concentration after CWI was delayed and TNF-α production was decreased, but IL-1β was not affected within 48 h after CWI. A delayed increase in neutrophil percentage and decrease in lymphocyte percentage occurred after CWI. Conclusion: These findings suggest that, even though CWI caused changes in stress and immune markers, the participants showed no predisposition to symptoms of the common cold within 48 h after CWI.

Bidirectional effects of IL-10+ regulatory B cells in Ldlr−/− mice

Limiting the pro-inflammatory immune response is critical for the treatment of atherosclerosis. Regulatory B cells (Bregs) can modulate the immune response through interleukin-10 (IL-10). Current data regarding Bregs and atherosclerosis is scarce and conflicting. In this study, we investigated the frequency of IL-10+ B cells during the development of atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr-/-) mice and studied the effect of adoptive transfer of IL-10+ B cells on atherosclerosis. We found a very strong inverse correlation between atherosclerosis severity and the frequency of IL-10+ B cells. This effect was cholesterol-independent and observed in spleen, draining lymph nodes and peritoneal cavity. To directly assess the effects of IL-10+ B cells on atherosclerosis, we expanded IL-10+ B cells ex vivo with anti-CD40 and selected pure and viable IL-10-secreting B cells and IL-10- B cells and adoptively transferred them to Ldlr-/- mice, respectively. While IL-10- B cells were strongly atherogenic compared to control-treated mice, IL-10+ B cells did not affect lesion size. Adoptive transfer of IL-10+ B cells strongly reduced circulating leukocyte numbers and inflammatory monocytes. In addition, they decreased CD4+ T cell activation and increased IL-10+ CD4+ T cell numbers. Interestingly, both IL-10+ and IL-10- B cells exacerbated serum cholesterol levels and resulted in fatty livers, which potentially masked the beneficial effects of IL-10+ B cells on atherosclerosis. These findings underscore the strong immune-regulating function of IL-10+ B cells and provide additional incentives to explore effective strategies that expand IL-10+ B cells in atherosclerosis.

Diurnal rhythms in peripheral blood immune cell numbers of domestic pigs

Diurnal rhythms within the immune system are considered important for immune competence. Until now, they were mostly studied in humans and rodents. However, as the domestic pig is regarded as suitable animal model and due to its importance in agriculture, this study aimed to characterize diurnal rhythmicity in porcine circulating leukocyte numbers. Eighteen pigs were studied over periods of up to 50h. Cosinor analyses revealed diurnal rhythms in cell numbers of most investigated immune cell populations in blood. Whereas T cell, dendritic cell, and eosinophil counts peaked during nighttime, NK cell and neutrophil counts peaked during daytime. Relative amplitudes of cell numbers in blood differed in T helper cell subtypes with distinctive differentiation states. Mixed model analyses revealed that plasma cortisol concentration was negatively associated with cell numbers of most leukocyte types, except for NK cells and neutrophils. The observed rhythms mainly resemble those found in humans and rodents.

A short-term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA-axis signaling in mice.

There is still uncertainty whether extremely low frequency electromagnetic fields (ELF‐EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF‐EMF for 1, 4, and 24 h/day in a short‐term (1 week) and long‐term (15 weeks) set‐up to investigate whole body effects on the level of stress regulation and immune response. ELF‐EMF signal contained multiple frequencies (20–5000 Hz) and a magnetic flux density of 10 μT. After exposure, blood was analyzed for leukocyte numbers (short‐term and long‐term) and adrenocorticotropic hormone concentration (short‐term only). Furthermore, in the short‐term experiment, stress‐related parameters, corticotropin‐releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene‐expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short‐term but not long‐term experiment, leukocyte counts were significantly higher in the 24 h‐exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short‐term ELF‐EMF exposure may affect hypothalamic‐pituitary‐adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433–443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Impact of melatonin receptor deletion on intracellular signaling in spleen cells of mice after polymicrobial sepsis.

Melatonin is known to influence immune functions and to ameliorate outcome after septic challenge but it is unknown whether this is mediated by melatonin receptor activation. This study aimed to elucidate molecular differences in spleen and ex vivo splenocytes of wild-type (WT) and melatonin receptor double knockout mice (KO) after polymicrobial sepsis. C3H/HeN wild-type and MT1-/-/MT2-/- mice underwent sham operation or cecum ligation and incision (CLI) and remained anesthetized for 1 h. Splenocytes were isolated and treated in culture with physiological melatonin concentrations (1 nM). Plasma TNFα levels were consistently high after 1 h of CLI. Basal circulating leukocyte numbers were slightly higher in KO animals. We detected transcriptional differences in splenocytes of the knockout strain concerning proinflammatory mediators. Expression levels of IL-1β, IL-2, CXCR2, L-Selectin, TNFα, CXCL2 and ICAM-1 were strongly increased in splenocytes of KO mice. Splenocytes of KO mice displayed reduced ERK and p38 as well as increased JNK phosphorylation. None of the analyzed factors were influenced by melatonin in the culture medium. The results of this study indicate an increased proinflammatory status of mice deficient in both membrane-bound melatonin receptors reflected by altered activation of MAPK cascades and transcriptional activation of proinflammatory mediators.

Rac-Null Leukocytes Are Associated with Increased Inflammation-Mediated Alveolar Bone Loss

Periodontitis is characterized by altered host-biofilm interactions that result in irreversible inflammation-mediated alveolar bone loss. Genetic and epigenetic factors that predispose to ineffective control of biofilm composition and maintenance of tissue homeostasis are not fully understood. We elucidated how leukocytes affect the course of periodontitis in Rac-null mice. Mouse models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to loss of alveolar bone due to inflammation in Rac-null mice. Leukocyte margination was differentially impaired in these mice during attachment in conditional Rac1-null (granulocyte/monocyte lineage) mice and during rolling and attachment in Rac2-null (all blood cells) mice. Inflammatory responses to subgingival ligatures, assessed by changes in peripheral blood differential leukocyte numbers, were altered in Rac-null compared with wild-type mice. In response to persistent subgingival ligature-mediated challenge, Rac-null mice had increased loss of alveolar bone with patterns of resorption characteristic of aggressive forms of periodontitis. These findings were partially explained by higher osteoclastic coverage of the bone-periodontal ligament interface in Rac-null compared with wild-type mice. In conclusion, this study demonstrates that leukocyte defects, such asdecreased endothelial margination and tissue recruitment, are rate-limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

Assessment of inflammatory resilience in healthy subjects using dietary lipid and glucose challenges

BackgroundResilience or the ability of our body to cope with daily-life challenges has been proposed as a new definition of health, with restoration of homeostasis as target resultant of various physiological stress responses. Challenge models may thus be a sensitive measure to study the body’s health. The objective of this study was to select a dietary challenge model for the assessment of inflammatory resilience. Meals are a challenge to metabolic homeostasis and are suggested to affect inflammatory pathways, yet data in literature are limited and inconsistent.MethodThe kinetic responses of three different dietary challenges and a water control challenge were assessed on various metabolic and inflammatory markers in 14 healthy males and females using a full cross-over study design. The dietary challenges included glucose (75 g glucose in 300 ml water), lipids (200 ml whipping cream) and a mix of glucose and lipids (same amounts as above), respectively. Blood samples were collected at baseline and at 0.5, 1, 2, 4, 6, 8 and 10 h after consumption of the treatment products. Inflammation (IFNγ, IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α CRP, ICAM-1, VCAM-1, SAA, E-selectin, P-selectin, thrombomodulin, leukocytes, neutrophils, lymphocytes) and clinical (e.g. glucose, insulin, triglycerides) markers as well as gene expression in blood cells and plasma oxylipin profiles were measured.ResultsAll three dietary challenges induced changes related to metabolic control such as increases in glucose and insulin after the glucose challenge and increases in triglycerides after the lipid challenge. In addition, differences between the challenges were observed for precursor oxylipins and some downstream metabolites including DiHETrE’s and HODE’s. However, none of the dietary challenges induced an acute inflammatory response, except for a modest increase in circulating leukocyte numbers after the glucose and mix challenges. Furthermore, subtle, yet statistically significant increases in vascular inflammatory markers (sICAM-1 and sVCAM-1) were found after the mix challenge, when compared to the water control challenge.ConclusionsThis study shows that dietary glucose and lipid challenges did not induce a strong acute inflammatory response in healthy subjects, as quantified by an accurate and broad panel of parameters.

Low dose LPS does not increase TLR4 expression on monocytes in a human in vivo model

Toll like receptor 4 (TLR4) is the major recognition receptor for lipopolysaccharides and plays a major role in the inflammatory response. CD11b is expressed on the surface of many leukocytes including monocytes. The CD11b/CD18 complex is involved in the inflammatory response by mediating migration and adhesion of leukocytes. The aim of this human in vivo study was to investigate the expression of TLR4 and CD11b on the surface of human monocytes after in vivo low-dose LPS stimulation. We performed a double-blind, randomized crossover study with 16 healthy males who received a bolus injection of bacterial lipopolysaccharide (LPS; 0.4ng/kg) or normal saline. Vital parameters, blood counts, serum cytokine levels, the expression of TLR4, and CD11b on CD14 positive cells were analyzed. The experimentally induced inflammatory response was reflected by transient increases in body temperature, circulating leukocyte numbers, and plasma levels of pro- (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10, IL-1ra). In contrast to a significant increase in CD11b expression, no changes in TLR4 expression on circulating monocytes were detectable. Early changes in TLR4 expression on circulating monocytes are not necessarily part of the inflammatory response to low dose LPS in humans whereas the detected increase of CD11b expression might already be sufficient for optimized recognition and signalling.

CBL-B is required for leukemogenesis mediated by BCR-ABL through negative regulation of bone marrow homing

BCR-ABL induces chronic myeloid leukemia (CML) through the aberrant regulation of multiple signaling substrates. Previous research has shown that BCR-ABL mediates down-modulation of CBL-B protein levels. A murine bone marrow transplantation (BMT) study was performed to assess the contribution of Cbl-b to BCR-ABL-induced disease. The predominant phenotype in the Cbl-b(-/-) recipients was a CML-like myeloproliferative disease (MPD) similar to that observed in the wild-type animals, but with a longer latency, diminished circulating leukocyte numbers and reduced spleen weights. Despite the decreased leukemic burden in comparison to their wild-type counterparts, the Cbl-b(-/-) animals displayed enhanced numbers of Gr-1(+)/Mac-1(+) spleen cells and neutrophilia. On the basis of prior evidence of CBL-B-dependent motility toward SDF-1α, we hypothesized that Cbl-b deficiency might impair bone marrow localization during transplantation. Homing experiments showed reduced migration of Cbl-b(-/-) cells to the bone marrow. Intrafemoral transplantation of BCR-ABL-transduced Cbl-b(-/-) cells revealed equivalent latency of disease development to the wild-type transplants, supporting the conclusion that Cbl-b deficiency diminishes homing of leukemic cells to the bone marrow, and perturbs the proliferation of BCR-ABL-expressing malignant clones during CML development.

Increased circulating leukocyte numbers and altered macrophage phenotype correlate with the altered immune response to brain injury in metallothionein (MT)-I/II null mutant mice.

BackgroundMetallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system.MethodsWild type and MT-I/II-/- mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay.ResultsComparison of MT-I/II-/- mice with wild type controls following cryolesion brain injury revealed that the MT-I/II-/- mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II-/- mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II-/- mice that was independent of brain injury.ConclusionsThese results contribute to the evidence that MT-I/II-/- mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II-/- mice after brain injury relative to wild type mice.

Leukocyte recruitment to the cremaster muscle exhibits circadian oscillations

Circulating leukocyte numbers exhibit circadian oscillations with peak values in mice during the day [Zeitgeber time, ZT 5 (hours after the onset of light)], and trough at night [ZT 13]. Here, we report that leukocyte recruitment to the cremaster muscle oscillates in antiphase with blood count patterns, with numbers of adherent and extravasated leukocytes peaking at night and exhibiting a nadir during the day (ZT5/ZT13: 5.3 ± 0.3/7.3 ± 0.3 cells per 100 × 50 μm2 extravascular vessel segment, P<0.001) as assessed by in vivo and ex vivo imaging of the cremasteric microvasculature. Circadian differences depended on ICAM‐1 expression and local innervation, as ICAM‐1 deficiency and surgical denervation of the genitofemoral nerve ablated oscillations. Systemic stimulation of mice with fMLP resulted in increased recruitment of granulocytes during the day compared to night (ZT5/ZT13: 1.3 ± 0.3/0.6 ± 0.3 cells per 100 × 50 μm2 extravascular vessel segment, P<0.05). On the other hand, mice subjected to a 12h jetlag did not exhibit circadian oscillations in leukocyte recruitment and failed to show inflammatory leukocyte infiltration after fMLP treatment. These data show that leukocyte trafficking to the cremaster exhibits circadian oscillations and suggest that circadian time can influence the recruitment of leukocytes during inflammation.Funded by the German Academic Exchange Service (DAAD) and the NIH.

Immune Response to Resistance Exercise

Resistance exercise produces transient perturbations in immunity, including alterations in circulating leukocyte numbers, cytokine concentration, and some measures of cell function. These changes are typically interpreted as being transiently detrimental to host defense. The mechanisms responsible for these immune fluctuations appear to be neuroendocrine-mediated alterations in cell trafficking and function and microtrauma-mediated alterations in cytokine release. Alterations in immunity following resistance exercise appear to be similar in pattern but smaller in magnitude than those typically seen after long, vigorous endurance exercise and are resolved within a few hours. However, resistance exercise—induced changes in immunity may become clinically relevant after repeated exercise bouts with insufficient recovery. Regular training appears to attenuate the immune response to resistance exercise. Care should be taken to ensure that resistance training is planned, with adequate variation in intensity and volume over time, to ensure recovery between sessions and to avoid chronic systemic inflammation.

Abstract 5201: Low-dose Tetrahydrobiopterin Supplementation Reduces Atherosclerosis Progression in Apolipoprotein E-Knockout Mice via Effects on Vascular T cell Infiltration

Tetrahydrobiopterin (BH4) supplementation improves endothelial function in models of vascular disease by maintaining endothelial nitric oxide synthase coupling and nitric oxide bioavailability. Recent studies using pharmacological and transgenic endothelial-specific BH4 supplementation highlighted the therapeutic potential of BH4 in native atherosclerosis and vascular injury and suggested a potential role for BH4 as regulator of inflammation and vascular remodeling. However, the cellular mechanisms through which this leads to reduced atherosclerosis remain unclear. We have used a pharmaceutical BH4 formulation to address the hypothesis that improved endothelial function resulting from exogenous BH4 supplementation leads to reduced atherosclerosis through effects on vascular inflammation. Single oral dose pharmacokinetic studies revealed rapid BH4 uptake from the gastrointestinal tract into plasma and organs. While in most organs BH4 levels returned to baseline by 8 hours after dosing, BH4 levels in aorta remained markedly increased at 24 hours, suggesting an active mechanism for BH4 retention. Daily oral BH4 supplementation in 8 week old chow-fed ApoE-KO mice for 8 or 12 weeks had no effect on plasma lipids or hemodynamic parameters, but resulted in a significant 45% relative reduction in aortic root atherosclerosis only at 10 mg/kg BH4, compared with placebo controls (0.049 vs. 0.088 mm2, P<0.05 after 8 weeks; 0.142 vs. 0.259 mm2, P<0.05 after 12 weeks). BH4 treatment led to a striking reduction in T cell infiltration, both within plaques (0.001886 vs. 0.006653 mm2; P<0.05) and in the surrounding adventitia (0.007741 vs. 0.020133 mm2; P<0.05). Importantly, BH4 treatment had no effect on circulating leukocyte numbers, and furthermore no evidence for BH4 uptake by peripheral blood mononuclear cells from plasma after single oral supplementation was found. Experiments using endothelium-targeted GTP-cyclohydrolase 1 transgenic mice, with a specific increase in endothelial BH4 levels, revealed a similar reduction in vascular T cell infiltration, suggesting that BH4 supplementation reduces atherosclerosis through a reduction in vascular T cell infiltration mediated by salutary effects on endothelial nitric oxide bioavailability.

Effects of chytridiomycosis on circulating white blood cell distributions of bullfrog larvae (Rana catesbeiana)

Bullfrogs (Rana catesbeiana) are widely believed to be nonclinical carriers of Batrachochytrium dendrobatidis (Bd), the fungal pathogen that invades keratinized tissues of amphibians and causes the disease, chytridiomycosis. Although most research on this disease focuses on adults, larval anurans are also susceptible to infections in their keratinized mouthparts, and this allows for visual diagnosis of the disease via the degree of mouthpart depigmentation. When an unplanned outbreak of chytridiomycosis occurred in a set of captive bullfrog tadpoles in our lab, we conducted the current investigation into its effects on the nonspecific immune system (i.e., the leukocyte populations) of the tadpoles. We compared leukocyte counts from blood smears of 27 tadpoles that had contracted the disease (evidenced by severe mouthpart depigmentation and confirmed by histology) to those of 21 tadpoles that had little depigmentation (i.e., with little evidence of the disease). Tadpoles with severe depigmentation had significantly more neutrophils and less eosinophils than those with little depigmentation, while numbers of lymphocytes, basophils, and monocytes were not statistically different. That there was any effect at all on circulating leukocyte numbers is surprising since leukocytes are usually not seen migrating to sites of infection in tissue sections of amphibians infected with Bd, and since most research points to this disease having little outward effect on bullfrogs. Since monocyte numbers were unchanged, the leukocyte alterations were likely not due to a simple inflammation response. It is possible that Bd infections elicit increases in glucocorticoid hormones, which can cause increased numbers of circulating neutrophils and lower numbers of eosinophils, although this is often accompanied by a reduction in lymphocyte numbers, which we did not see. Further research is warranted to clarify if this effect is limited to this species.