Einfluss des Chemokinrezeptors 4 (CXCR4) und seiner Liganden auf die systemische Inflammation

Aufgrund der hohen Mortalität der Sepsis ist die Erforschung kausaler Therapien weiterhin von entscheidender Bedeutung. Im Fokus der vorliegenden Arbeit stand daher die Untersuchung des Einflusses des Chemokinrezeptors CXCR4 auf eine schwere systemische Entzündungsreaktion. Zunächst wurden in einem Vorversuch sowohl verschiedene Mausmodelle für eine systemische Inflammation als auch der ideale Zeitpunkt für die geplante nachfolgende Untersuchung der Effekte von CXCR4-Liganden auf die systemische Entzündung ermittelt. Dabei zeigte sich das Verabreichen von Lipopolysacchariden (LPS) und der Zeitpunkt 24 Stunden nach LPS-Gabe am besten dazu geeignet, eine potentiell anti-inflammatorische Substanz zu testen. Nachfolgend wurde der CXCR4 zunächst mit dem spezifischen Antagonisten AMD3100 blockiert und die Effekte auf die LPS-induzierten Veränderungen zahlreicher systemischer und Organfunktionsparameter erfasst. Es zeigte sich eine verstärkte Entzündungsreaktion gegenüber der alleinigen LPS-Gabe. Dies war vor allem an einem verschlechterten Allgemeinzustand, erhöhten Serumzytokinspiegeln, einer verminderten Biotransformationskapazität und an vermehrt in die Organe immigrierten Immunzellen zu erkennen. Entsprechend wurde daraufhin ein plasmastabiler CXCR4-Agonist untersucht. Dieser verbesserte das Krankheitsbild deutlich im Vergleich zur alleinigen LPS-Gabe, was an nahezu allen ermittelten Parametern zu erkennen war. Der Allgemeinzustand verbesserte sich, die Serumzytokinspiegel und der oxidative Stress in den Organen wurden verringert und die Biotransformationskapazität nahm zu. Weiterhin zeigten sich eine verminderte Immunzellumverteilung und eine deutliche Induktion des protektiven Enzym Hämoxygenase-1, was maßgeblich zu dem verbesserten Allgemeinzustand beigetragen haben könnte. Alles in allem lässt sich festhalten, dass die Blockade des CXCR4 die systemische Entzündungsreaktion verstärkt, wohingegen die Aktivierung des Rezeptors das Krankheitsbild verbessert

Administration of AMD3100 in endotoxemia is associated with pro-inflammatory, pro-oxidative, and pro-apoptotic effects in vivo

BACKGROUND: Chemokine receptor 4 (CXCR4) is a multifunctional G protein-coupled receptor that is activated by its natural ligand, C-X-C motif chemokine 12 (CXCL12). As a likely member of the lipopolysaccharide (LPS)-sensing complex, CXCR4 is involved in pro-inflammatory cytokine production and exhibits substantial chemo-attractive activity for various inflammatory cells. Here, we aimed to characterize the effects of CXCR4 blockade in systemic inflammation and to evaluate its impact on organ function. Furthermore, we investigated whether CXCR4 blockade exerts deleterious effects, thereby substantiating previous studies showing a beneficial outcome after treatment with CXCR4 agonists in endotoxemia. METHODS: The CXCR4 antagonist AMD3100 was administered intraperitoneally to mice shortly after LPS treatment. After 24 h, health status was determined and serum tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma), and nitric oxide (NO) levels were measured. We further assessed oxidative stress in the brain, kidney, and liver as well as liver biotransformation capacity. Finally, we utilized immunohistochemistry and immunoblotting in liver and spleen tissue to determine cluster of differentiation 3 (CD3), CD8, CD68, and TNF alpha expression patterns, and to assess the presence of various markers for apoptosis and oxidative stress. RESULTS: Mice treated with AMD3100 displayed impaired health status and showed enhanced serum levels of TNF alpha, IFN gamma and NO levels in endotoxemia. This compound also amplified LPS-induced oxidative stress in all tissues investigated and decreased liver biotransformation capacity in co-treated animals. Co-treatment with AMD3100 further inhibited expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), heme oxygenase-1 (HO-1), and various cytochrome P450 enzymes, whereas it enhanced expression of CD3, inducible nitric oxide synthase, and TNF alpha, as well as the total number of neutrophils in liver tissue. Spleens from co-treated animals contained large numbers of erythrocytes and neutrophils, but fewer CD3+ cells, and demonstrated increased apoptosis in the white pulp. CONCLUSIONS: AMD3100 administration in a mouse model of endotoxemia further impaired health status and liver function and mediated pro-inflammatory, pro-oxidative, and pro-apoptotic effects. This suggests that interruption of the CXCR4/CXCL12 axis is deleterious in acute inflammation and confirms previous findings showing beneficial effects of CXCR4 agonists in endotoxemia, thereby more clearly elucidating the role of CXCR4 in inflammation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12929-016-0286-8) contains supplementary material, which is available to authorized users

Administration of a CXCL12 Analog in Endotoxemia Is Associated with Anti-Inflammatory, Anti-Oxidative and Cytoprotective Effects <i>In Vivo</i>

<div><p>Background</p><p>The chemokine receptor CXCR4 is a multifunctional receptor which is activated by its natural ligand C-X-C motif chemokine 12 (CXCL12). As CXCR4 is part of the lipopolysaccharide sensing complex and CXCL12 analogs are not well characterized in inflammation, we aimed to uncover the systemic effects of a CXCL12 analog in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by using a sublethal LPS dose.</p><p>Methods</p><p>The plasma stable CXCL12 analog CTCE-0214D was synthesized and administered subcutaneously shortly before LPS treatment. After 24 hours, mice were sacrificed and blood was obtained for TNF alpha, IFN gamma and blood glucose evaluation. Oxidative stress in the liver and spleen was assessed and liver biotransformation capacity was determined. Finally, CXCR4, CXCL12 and TLR4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for apoptosis and oxidative stress were determined by means of immunohistochemistry.</p><p>Results</p><p>CTCE-0214D distinctly reduced the LPS mediated effects on TNF alpha, IFN gamma, ALAT and blood glucose levels. It attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. Furthermore, in all three organs investigated, CTCE-0214D diminished the LPS induced expression of CXCR4, CXCL12, TLR4, NF-κB, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity was induced above average. Additionally, TUNEL staining revealed anti-apoptotic effects of CTCE-0214D.</p><p>Conclusions</p><p>In summary, CTCE-0214D displayed anti-inflammatory, anti-oxidative and cytoprotective features. It attenuated reactive oxygen species, induced heme oxygenase 1 activity and mitigated apoptosis. Thus, the CXCR4/CXCL12 axis seems to be a promising target in the treatment of acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals.</p></div

Periodic acid-Schiff (PAS) staining and immunohistochemical staining of heme oxygenase 1 in the livers.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: (A-C) 200x, (D-G) 400x, (H) 630x). For reasons of clarity, the PAS staining of the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control. Exposure to LPS (B) evoked an impressive loss of glycogen content in the livers, whereas the livers of CTCE-0214D plus LPS treated mice (C) showed no difference to the control group (A) at all. LPS exposure (E) enhanced HO-1 occurrence in Kupffer and pit cells, whereas CTCE-0214D on its own (F) was able to induce HO-1 above average. Combined CTCE-0214D plus LPS administration (G) resulted in maximum HO-1 levels. The photomicrograph in (H) shows single Kupffer and pit cells, respectively, at a higher magnification (630x). To quantify the HO-1 activity in the liver, we performed an assay in the 9000g supernatants (I). LPS, CTCE-0214D and CTCE-0214D plus LPS administration increased the HO-1 activity by approximately 90%, 120% and 210%, respectively, when compared to the control group. The combined CTCE-0214D plus LPS treatment revealed the highest HO-1 activity levels, which were additionally significantly elevated in comparison to the values of the LPS or CTCE-0214D group, respectively. Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001 vs. control; ⁺, p≤0.05; ⁺⁺, p≤0.01; ⁺⁺⁺, p≤0.001 vs. LPS; #, p≤0.05; ##, p≤0.01; ###, p≤0.001 vs. CTCE-0214D.</p

Additional file 1: of Administration of AMD3100 in endotoxemia is associated with pro-inflammatory, pro-oxidative, and pro-apoptotic effects in vivo

Protein content in the liver, serum creatinine and urea levels and TNF alpha assay in THP-1 monocytes as well as macrophages. C57BL/6 N mice were treated either with LPS (5 mg/kg body weight), AMD3100 (5 mg/kg body weight), with both substances or with the solvent PBS (control). 24 h thereafter, the mice were sacrificed and the livers were collected for the determination of the protein content in the 9000 g supernatants by using the Biuret method (A). Also, the blood was obtained and serum creatinine as well as serum urea levels were measured (B, C). Data are given as mean ± standard error of the mean (SEM); n = 7 for each group. Statistical significant differences between the different treatment groups were determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test and are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control animals; +, p < 0.05; ++, p < 0.01; +++, p < 0.001 vs. LPS treatment. THP-1 cells were cultured in RPMI 1640 supplemented with 10 % fetal bovine serum and 100 nM penicillin/streptomycin (all Capricorn Scientific, Ebsdorfergrund, Germany) and were cultured at a density of about 1.0 × 106/ml at 5 % CO2 at 37 °C. To obtain THP-1 macrophages, cells were differentiated with PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich, St. Louis, MO) at a concentration of 40 ng/ml for three days. For the experiments, the cells were pretreated with AMD3100 or CXCL12, respectively, 30 min before LPS was added. After additional 12 h, the media were collected and analyzed for TNF alpha concentration by using a TNF alpha ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA) (D, E). Data are given as mean ± standard error of the mean (SEM); n = 3 independent experiments. (PDF 169 kb

Immunohistochemical staining of TLR4, NF-κB and cleaved caspase-3 in the spleens.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: (A-I) 200x, (J) 630x). For reasons of clarity, the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control. TLR4 occurred primarily in the white pulp, whereas cells of the red pulp displayed almost no membrane-bound staining. Especially after LPS treatment (B), spleno- and lymphocytes were identified to show an intensive TLR4 expression. Co-administration of CTCE-0214D to LPS clearly decreased the staining intensity of NF-κB, as lympho—and splenocytes in lymphoid follicles of the white pulp displayed diminished NF-κB levels (F). Nevertheless, the nuclear factor occurred ubiquitously in the spleens of all groups. Besides a few spleno- and lymphocytes, especially tingible body macrophages showed advanced cleaved caspase-3 activity and were found unambiguously more often in LPS-challenged mice (H,J). Co-administration of CTCE-0214D plus LPS reduced the appearance of cleaved caspase-3 to a minimum (I).</p

Comprehensive comparison of three different animal models for systemic inflammation

Abstract Background To mimic systemic inflammation in humans, different animal models have been developed. Since these models are still discussed controversially, we aimed to comparatively evaluate the most widely used models with respect to the systemic effects, the influence on organ functions and to the underlying pathophysiological processes. Methods Systemic inflammation was induced in C57BL/6N mice with lipopolysaccharide (LPS) treatment, peritoneal contamination and infection (PCI), or cecal ligation and puncture (CLP). Blood glucose and circulating cytokine levels were evaluated at 0, 2, 4, 6, 12, 24, 48, and 72 h after induction of inflammation. Additionally, oxidative stress in various organs and liver biotransformation capacity were determined. Markers for oxidative stress, apoptosis, infiltrating immune cells, as well as cytokine expression patterns, were assessed in liver and spleen tissue by immunohistochemistry. Results Treating mice with LPS and PCI induced a very similar course of inflammation; however, LPS treatment elicited a stronger response. In both models, serum pro-inflammatory cytokine levels rapidly increased whereas blood glucose decreased. Organs showed early signs of oxidative stress, and apoptosis was increased in splenic cells. In addition, liver biotransformation capacity was reduced and there was pronounced immune cell infiltration in both the liver and spleen. Mice exposed to either LPS or PCI recovered after 72 h. In contrast, CLP treatment induced comparatively fewer effects, but a more protracted course of inflammation. Conclusions The LPS model of systemic inflammation revealed to be most suitable when being interested in the impact of new therapies for acute inflammation. When using the CLP model to mimic human sepsis more closely, a longer time course should be employed, as the treatment induces delayed development of systemic inflammation

TUNEL assay in the spleens and immunohistochemical staining of the cleaved caspase-3 in the thymi.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: (A-C) 400x, (D-F) 200x, (G,H) 630x). For reasons of clarity, the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control group. Whereas endotoxin induced cell death in both the white and red pulp (B), CTCE-0214D was able to significantly diminish the amount of cells undergoing apoptosis (C), which is particularly evident in the white pulp. In the thymi of the LPS group, endotoxin caused a massive appearance of cleaved caspase-3 in the cortex (E), whereas the medulla did not significantly differ from the other treatment groups. CTCE-0214D decreased the cleaved caspase-3 expression in the cortical regions to control level and caused a raise in the amount of lymphocytes (F), especially when compared to the LPS group. The magnifications in (G) and (H) reveal the tingible body macrophages in the white pulp to be the main locus of apoptotic cell death.</p

Oxidative stress in the liver and spleen.

<p>Tissue content of lipid peroxidation products as determined by thiobarbituric acid reactive substances (TBARS) in the livers of all animal groups 24 hours after treatment (A). CTCE-0214D reduced TBARS elevated due to endotoxemia to almost the half. As further parameters, total glutathione content (B), reduced glutathione levels (GSH, C) and the GSH/GSSG ratio were evaluated (D). In comparison to the control group, endotoxin caused a decrease in the total as well as reduced glutathione content and minimized the GSH/GSSG ratio, while co-administration of CTCE-0214D was able to mitigate all LPS effects significantly. Endotoxin caused a significantly reduced GSH/GSSG ratio in the spleen, whereas CTCE-0214D was able to increase the ratio which was attributable mainly to reduced GSSG levels. Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001.</p

Biotransformation capacity.

<p>Benzyloxyresorufin-O-debenzylation [BROD] (A), ethoxyresorufin-O-deethylation [EROD] (B), ethylmorphine-N-demethylation [EMND] (C) and glutathione-S-transferase (D, as 1-chloro-2,4-dinitrobenzene conjugation) activities in 9000g supernatants are shown exemplarily. LPS impaired BROD activities to approximately 40% of the control values, whereas enzyme activities of CTCE-0214D plus LPS treated mice were elevated by more than 90% when compared to endotoxin administration alone. Moreover, no significant difference was detectable between CTCE-0214D plus LPS or saline treated mice. Furthermore, CTCE-0214D plus LPS treated mice revealed significantly elevated EROD activities when compared to LPS treatment, whereas no significance to the control group was detectable. Co-administration of CTCE-0214D and LPS ameliorated endotoxins effects on EMND activities (elevation by about 70%), the activity, however, still being significantly decreased when compared to control. Furthermore, increased glutathione-S-transferase activities were observed when comparing the CTCE-0214D plus LPS to the LPS group. Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001. In (E), the results of all performed model reactions are presented in pmol/(mg protein x min), except PNPH and GST, which are given in nmol/(mg protein x min). Statistical significance was determined and marked as mentioned, with the addition that significance to the LPS group was marked separately; ⁺, p≤0.05; ⁺⁺, p≤0.01; ⁺⁺⁺, p≤0.001 vs. LPS.</p