Nanobody-loaded immunosorbent for highly-specific removal of interleukin-17A from blood.

Elimination of overproduced cytokines from blood can relieve immune system disorders caused by hypercytokinemia. Due to the central roles of interleukin-17A (IL-17A) plays in regulating the immunity and inflammatory responses in humans, here, a novel immunosorbent containing anti-IL-17A nanobodies (Nbs) was constructed for IL-17A removal from blood. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm is up to 11.55 mg/g gel, which is almost consistent with the saturated adsorption capacity determined in dynamic adsorption. The in vitro plasma perfusion test demonstrated a remarkable adsorptive performance of the Nb-coupled sorbent since more than 75% IL-17A could be eliminated under the plasma/sorbent ratio of 1000:1. These results indicated the Nb-loaded immunosorbent can provide a simple and economic platform technology for immunoaffinity depletion of single or even multiple cytokines from plasma.

Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris.

Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.

[Eukaryotic expression and biological activity of recombinant human IL-17B protein].

AIM: To construct pCEP4/hIL-17B recombinant expression vector and express it stably in eukaryotic cells and investigate the biological activity in vitro. METHODS: The CDS region of human IL-17B gene was cloned by RT-PCR. After identification by sequencing, the hIL-17B gene was inserted into expression vector of pCEP4 to construct the recombinant vector pCEP4/hIL-17B, then transfected into 293T cells. The transgenic 293T cell line stably expressing rhIL-17B protein was selected in the presence of Hygromycin B. After FCS-free cultivation and sub-cloning, The IL-17B/mFc gene and protein expression was confirmed by RT-PCR, ELISA and Western blot analysis. To investigate the ability of combination with IL-17B receptor on human leukemic monocytic cell line, THP-1, by Flow cytometrical analysis (FACS) and of stimulation to secret cytokines in vitro. RESULTS: The recombinant pCEP4/hIL-17B and its transgenic 293T cells stably expressing rhIL-17B protein were obtained successfully. FACS analysis showed its high affinity with its receptor and it can stimulated THP-1 cell line to excrete IL-1ß and TNF-α in vitro and consistently caused a dose-dependent influx of neutrophil into the peritoneal cavity by intraperitoneal injection in vivo. CONCLUSION: The obtainment of transgenic 293T cell line stably expressing rhIL-17B protein paved the way for further study on biological functions of hIL-17B.

[Prokaryotic expression and biological activity of recombinant human IL-17F/His protein].

AIM: To Prepare recombinant human IL-17F/His protein and investigate its biological activity in vitro. METHODS: The gene region of human IL-17F was cloned by RT-PCR. After identification by sequencing, the hIL-17F gene encoding function domain was cloned into expression plasmid PQE3.0 and transfected into E.coli M15. By the induction of Isopropyl-ß-D-Thiogalacto-Pyranoside(IPTG), recombinant IL-17F/His protein was effectively expressed in E.coli M15. The recombinant protein was identified by Western blot. RESULTS: After renaturation and purification by HiTrap(TM); affinity column, the recombinant protein can up-regulate macrophages to secret TNF-α, IL-6 and other relative cytokines. It also promoted proliferation of HeLa cells in vitro. CONCLUSION: hIL-17F/His recombinant protein is of high biological activity, which can be used to make further study of its special characteristic.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

Expression, refolding and purification of a human interleukin-17A variant.

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.

Cutting edge: IL-17D, a novel member of the IL-17 family, stimulates cytokine production and inhibits hemopoiesis.

A novel cytokine termed IL-17D was cloned using nested RACE PCR. It is a secreted cytokine with homology to the IL-17 family of proteins. IL-17D is preferentially expressed in skeletal muscle, brain, adipose tissue, heart, lung, and pancreas. Treatment of endothelial cells with purified rIL-17D protein stimulated the production of IL-6, IL-8, and GM-CSF. The increased expression of IL-8 was found to be NF-kappa B-dependent. rIL-17D also demonstrated an inhibitory effect on hemopoiesis of myeloid progenitor cells in colony formation assays.

Forced expression of murine IL-17E induces growth retardation, jaundice, a Th2-biased response, and multiorgan inflammation in mice.

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions. In this study, we have identified the murine ortholog of IL-17E and developed transgenic mice to characterize its actions in vivo. Biological consequences of overexpression of murine (m)IL-17E, both unique to IL-17E and similar to IL-17, were revealed. Exposure to mIL-17E resulted in a Th2-biased response, characterized by eosinophilia, increased serum IgE and IgG1, and a Th2 cytokine profile including elevated serum levels of IL-13 and IL-5 and elevated gene expression of IL-4, IL-5, IL-10, and IL-13 was observed in many tissues. Increased gene expression of IFN-gamma in several tissues and elevated serum TNF-alpha were also noted. In addition, IL-17E induces G-CSF production in vitro and mIL-17E-transgenic mice had increased serum G-CSF and exhibit neutrophilia, a property shared by IL-17. Moreover, exposure to mIL-17E elicited pathological changes in multiple tissues, particularly liver, heart, and lungs, characterized by mixed inflammatory cell infiltration, epithelial hyperplasia, and hypertrophy. Taken together, these findings suggest that IL-17E is a unique pleiotropic cytokine and may be an important mediator of inflammatory and immune responses.

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma.

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4(+) T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 +/- 19.1 pg/ml; for IL-8, 1724.2 +/- 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 +/- 55.6 pg/ml; for IL-8, 4371.4 +/- 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 +/- 4.39 vs baseline, MFI = 12.26 +/- 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 +/- 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.

IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines.

BACKGROUND: IL-17 is a cytokine that has been reported to be produced by T lymphocytes. In vitro, IL-17 activates fibro-blasts and macrophages for the secretion of GM-CSF, TNF-alpha, IL-1beta, and IL-6. A number of these cytokines are involved in the airway remodeling that is observed within the lungs of asthmatic individuals. OBJECTIVE: In this study, we investigated the expression of IL-17 in sputum and bronchoalveolar lavage specimens obtained from asthmatic subjects and from nonasthmatic control subjects. METHODS: IL-17 was detected through use of immunocytochemistry, in situ hybridization, and Western blot. Bronchial fibroblasts were stimulated with IL-17, and cytokine production and chemokine production were detected through use of ELISA and RT-PCR. RESULTS: Using immunocytochemistry, we demonstrated that the numbers of cells positive for IL-17 are significantly increased in sputum and bronchoalveolar lavage fluids of subjects with asthma in comparison with control subjects (P <.001 and P <.005, respectively). We demonstrated that in addition to T cells, eosinophils in sputum and bronchoalveolar lavage fluids expressed IL-17. Peripheral blood eosinophils were also positive for IL-17, and the level of IL-17 in eosinophils purified from peripheral blood was significantly higher in subjects with asthma than in controls (P <.01). To further investigate the mechanism of action of IL-17 in vivo, we examined the effect of this cytokine on fibroblasts isolated from bronchial biopsies of asthmatic and nonasthmatic subjects. IL-17 did enhance the production of pro-fibrotic cytokines (IL-6 and IL-11) by fibroblasts, and this was inhibited by dexamethasone. Similarly, IL-17 increased the level of other fibroblast-derived inflammatory mediators, such as the alpha-chemokines, IL-8, and growth-related oncogene-alpha. CONCLUSION: Our results, which demonstrate for the first time that eosinophils are a potential source of IL-17 within asthmatic airways, suggest that IL-17 might have the potential to amplify inflammatory responses through the release of proinflammatory mediators such as alpha-chemokines.

Cytokine expression and synovial pathology in the initiation and spontaneous resolution phases of adjuvant arthritis: interleukin-17 expression is upregulated in early disease.

The aim of this study was to understand the immune processes controlling the initiation and spontaneous resolution of adjuvant arthritis (AA). We investigated synovial T-cell recruitment and mRNA expression of IL-17 and other important disease related cytokines, IFN-gamma, IL-2, IL-4, TNF and TGF-beta in inguinal lymph node (ILN) and synovial membrane (SM). Arthritis severity was assessed by a numerical rating score and rats were sacrificed every 3--4 days postadjuvant induction. Further assessment involved quantitative radiology and histology of the ankle joints on each day, and the ILN and SM were removed for RNA extraction. Cytokine mRNA expression was measured using RT-PCR and densitometry. Paraffin sections of rat ankle joints were stained for T-cells (CD3) by immunohistochemistry. In the ILN, there was an increase in IL-17, TNF and IFN-gamma expression in the early stages of disease, with a secondary sustained increase in IFN-gamma expression. In the SM, there was expression of T-cell cytokines in early arthritis (day 13), and prolonged TNF and TGF-beta expression, which reflected disease progression. IL-4 mRNA expression increased in the later stages of AA. Synovial T-cell numbers transiently increased at day 6, and remained high from days 13--28. Increased pro-inflammatory cytokine expression, including IL-17, in the ILN reflects the initiating events in the early stage of disease. IL-17 may therefore play an important role in the pathogenesis of AA. The increase in IL-4 (an anti-inflammatory cytokine) in the SM in the later stages of AA suggests that IL-4 is involved in the spontaneous resolution of AA. The initial increase in IFN-gamma in the ILN may reflect a pro-inflammatory response, while the prolonged secondary increase may indicate activation of regulatory T-cells.