Level of inflammatory cytokines tumour necrosis factor α\alpha, interleukins 12, 23 and 17 in patients with psoriasis in the context of metabolic syndrome

Introduction: Psoriasis is a chronic inflammatory skin disease with immunologic etiology. Aim: To investigate the levels of the proinflammatory cytokines tumor necrosis factor \alpha ($TNF-\alpha$), interleukin 23 (IL-23) and IL-17 in patients with psoriasis and psoriatic arthritis with concomitant metabolic syndrome. Material and methods: This study included 60 patients with severe psoriasis. Results: In patients with arterial hypertension concomitant with psoriasis, no statistically significant differences in cytokine levels were observed. On the other hand, in the group of patients diagnosed with diabetes, an increased level of IL-17 was observed. In patients with lipid disorders, the results were similar to the results of patients with diabetes. Conclusions: It is very important to study immunologic mechanisms responsible for the presence and severity of psoriasis, in order to personalize the therapy in the future and optimize the effect of action on the basic disease and on concomitant disorders

Characteristics of the Alternative Phenotype of Microglia/Macrophages and its Modulation in Experimental Gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas

Influx of microglia/macrophages into the tumor is blocked by CsA.

<p>A. Representative confocal images of Iba1 staining in intact brain tissue, tumor-bearing brain slices from mice treated with PBS or CsA. Scale bar = 20 µm. B–C. Quantification of microglia and blood-derived macrophages in naïve, tumor-bearing and CsA-treated mice (4 per group). Each bar represents the mean ± SEM. ^***<i>p</i><0.001, ^**<i>p</i><0.01 tumor-bearing versus naïve mice; ^##<i>p</i><0.01, CsA-treated versus PBS-treated tumor-bearing mice.</p

Accumulation and activation of microglia/macrophages in experimental glioma.

<p>A. Representative confocal images of brain sections 15 days after implantation of pEGFP-N1 GL261 cells into the striatum of C57BL/6 mice. Note the infiltration and morphological transformation of glioma-infiltrating Iba1⁺ cells. Scale bar: left image – 1000 µm, right image – 20 µm. B. Contralateral and ipsilateral hemisphere from tumor-bearing brain 15 days after injection of pEGFP-N1 GL261 cells. Images showed merged Iba1 and EGFP fluorescence. Scale bar = 750 µm. C. Microglia/macrophages were separated using a magnetic-bead-conjugated anti-CD11b antibody and stained with CD45 PerCP-Cy5.5 and CD11b PE prior to FACS acquisition. Propidium iodide staining was performed to eliminate necrotic/apoptotic cells (Gate R3, R4) and viable cells were gated (Gate R1; <b>B1</b>, Gate R2; <b>B2</b>). D. Efficiency of CD11b-positive cells separation in the negative fraction (CD11b-negative cells) from each sample was controlled. E. Representative dot plots for microglia (Gate R4, CD11b⁺/CD45^low) and macrophages (Gate R5, CD11b⁺/CD45^high) from tumor-bearing hemispheres. F. Kinetics of microglia/macrophage influx into tumor tissue. CD11b⁺ cells separated from the brains of naïve, sham operated and tumor-bearing mice at day 3, 8 or 15 after implantation (n = 4/group) were sorted according to CD45 expression. Each bar represents the mean ± SEM; ^***<i>p</i><0.001, ^*<i>p</i><0.05 tumor-bearing mice at 8th day versus naïve mice; ^##<i>p</i><0.01 tumor-bearing mice at day 15 versus day 8.</p

Cytokine profile in glioma-bearing brains.

<p>A. The levels of ten pro/anti-inflammatory cytokines were determined by flow cytometry in protein extracts isolated from hemispheres of naïve, LPS-injected and tumor-bearing mice. The results show the means ± SEM (n = 5 per group); ^#<i>p</i><0.05 significant change between LPS-treated v. naïve mice; ^*<i>p</i><0.05 significant difference between tumor-bearing v. naïve mice. B. Elevated levels of IL-10 and GM-CSF detected in tumors are reduced by CsA treatment. Each dot represents an individual animal; a horizontal line represents a mean of each group; ^*<i>p</i><0.05; ^**<i>p</i><0.01. C. Expression of IL-10 in glioma-infiltrating microglia and macrophages. Left panel: expression of IL-10 on sorted CD11b⁺ cells (50,000 cells) determined by flow cytometry with the anti-IL-10 antibody conjugated to Alexa Fluor647. Representative histograms of IL-10 detection in microglia (light grey) and macrophages (dark grey) cells from naïve (a) and tumor-bearing (b) brains. Right panel: quantification of microglia/macrophages expressing IL-10 in naïve and tumor-bearing brains (means ± SEM, 3 animals per group); significant increase of IL-10-positive microglia (** <i>p</i><0.01) and macrophages (^###<i>p</i><0.001) in tumor-bearing brains. D. Quantification of <i>gm-csf</i> and <i>m-csf</i> expression in GL261 glioma cells and non-transformed astrocytes (means ± SD from 3 experiments). E. Quantitative evaluation of <i>gm-csf</i> mRNA expression in GL261 glioma cells exposed to 0.1 and 1 µM CsA for 24 hours (means ± SD from two experiments with three or four replicates per condition) compared to untreated control cells. Statistical analysis was done by Student <i>t</i> test, ** <i>p</i><0.01.</p

Quantification of selected M1/M2 phenotype-associated gene expression in CD11b⁺ cells isolated from naïve and tumor-bearing mice.

<p>Gene expression was analyzed by real-time PCR and the results are presented as fold changes of CD11b⁺ cells isolated from tumor brains versus those from naïve brain tissue. Numbers corresponding to the significantly changed genes (t-test generated p-value<0.05) are marked in bold; NA - not available.</p

Alterations of gene expression in infiltrating microglia/macrophages and intracranial gliomas are modulated by CsA.

<p>A. Gene expression in magnetically sorted CD11b⁺ cells from tumor-bearing and naïve brains was determined by qPCR. Expression of five genes was significantly altered in CD11b⁺ cells: <i>arg-1 (p = 0.000003)</i>; <i>cxcl14 (p = 0.0001)</i>; <i>ifn-β1 (p = 0.0002)</i>; <i>cox-2 (p = 0.000002)</i>; <i>mt1-mmp (p = 0.00002)</i>; n = 5 animals per group; ^*<i>p</i><0.05, ^**<i>p</i><0.01. The middle line represents the median value. Lower ΔC_T are consistent with higher gene expression. B. Quantification of arginase activity in brain tissue extracts from naïve and tumor-bearing mice treated either with PBS or CsA. Results represent the mean ± SEM of 4–5 mice; ^*<i>p</i><0.05, tumor-bearing versus tumor-free hemispheres; ^#<i>p</i><0.05, CsA (10 mg/kg, 8th) versus PBS-treated, tumor-bearing mice. C. MMP-2 activity in proteins extracts from the brains of naïve (N1–5) and tumor-bearing mice (T1–5) determined by gel zymography. Active MMP-2 detected as a prominent band at 62 kDa. D. Quantification of MMP-2 activity using the cleavage of fluorescent DQ-gelatin substrate; means ± SEM of 4–6 mice; ^**<i>p</i><0.01, tumor-bearing versus naïve brain extracts; ^###<i>p</i><0.001, ^#<i>p</i><0.05, CsA- versus PBS-treated tumor-bearing mice.</p