Macrophage-derived reactive oxygen species protects against autoimmune priming with a defined polymeric adjuvant.

Understanding the nature of adjuvants and the immune priming events in autoimmune diseases, such as rheumatoid arthritis, is a key challenge to identify their aetiology. Adjuvants are, however, complex structures with inflammatory and immune priming properties. Synthetic polymers provide a possibility to separate these functions and allow studies of the priming mechanisms in vivo. A well-balanced polymer, poly-N-isopropyl acrylamide (PNiPAAm) mixed with collagen type II (CII) induced relatively stronger autoimmunity and arthritis compared with more hydrophilic (polyacrylamide) or hydrophobic (poly-N-isopropylacrylamide-co-poly-N-tertbutylacrylamide and poly-N-tertbutylacrylamide) polymers. Clearly, all the synthesized polymers except the more hydrophobic poly-N-tertbutylacrylamide induced arthritis, especially in Ncf1-deficient mice, which are deficient in reactive oxygen species (ROS) production. We identified macrophages as the major infiltrating cells present at PNiPAAm-CII injection sites and demonstrate that ROS produced by the macrophages attenuated the immune response and the development of arthritis. Our results reveal that thermo-responsive polymers with high immune priming capacity could trigger an autoimmune response to CII and the subsequent arthritis development, in particular in the absence of NOX2 derived ROS. Importantly, ROS from macrophages protected against the autoimmune priming, demonstrating a critical regulatory role of macrophages in immune priming events.

Effects by periodontitis on pristane-induced arthritis in rats.

BACKGROUND: An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. METHODS: We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. RESULTS: Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. CONCLUSIONS: Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.

Collagen type II and a thermo-responsive polymer of N-isopropylacrylamide induce arthritis independent of Toll-like receptors: a strong influence by major histocompatibility complex class II and Ncf1 genes.

We established and characterized an arthritis mouse model using collagen type II (CII) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNiPAAm). The new PNiPAAm adjuvant is TLR-independent, as all immunized TLR including MyD88-deficient mice developed an anti-CII response. Unlike other adjuvants, PNiPPAm did not skew the cytokine response (IL-1ß, IFN-γ, IL-4, and IL-17), as there was no immune deviation towards any one type of immune spectrum after immunization with CII/PNiPPAm. Hence, using PNiPAAm, we studied the actual immune response to the self-protein, CII. We observed arthritis and autoimmunity development in several murine strains having different major histocompatibility complex (MHC) haplotypes after CII/PNiPAAm immunization but with a clear MHC association pattern. Interestingly, C57Bl/6 mice did not develop CII-induced arthritis, with PNiPAAm demonstrating absolute requirement for a classical adjuvant. Presence of a gene (Ncf1) mutation in the NADPH oxidation complex has a profound influence in arthritis and using PNiPAAm we could show that the high CIA severity in Ncf1 mutated mice is independent of any classical adjuvant. Macrophages, neutrophils, eosinophils, and osteoclasts but not mast cells dominated the inflamed joints. Furthermore, arthritis induction in the adjuvant-free, eosinophil-dependent Vß12 DBA/1 mice could be shown to develop arthritis independent of eosinophils using CII/PNiPAAm. Thus, biocompatible and biodegradable PNiPAAm offers unique opportunities to study actual autoimmunity independent of TLR and a particular cytokine phenotype profile.

Antibodies to a Citrullinated Porphyromonas gingivalis Epitope Are Increased in Early Rheumatoid Arthritis, and Can Be Produced by Gingival Tissue B Cells: Implications for a Bacterial Origin in RA Etiology.

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the presence of ACPA in RA patients, through mechanisms of molecular mimicry.

Polymeric cryogels are biocompatible, and their biodegradation is independent of oxidative radicals.

Biocompatibility and in vivo degradation are two important characteristics of cell scaffolds. We evaluated these properties for four different polymeric macroporous cryogels, polyvinylcaprolactam, polyvinyl alcohol-alginate-bioactive glass composite, polyhydroxyethylmethacrylate-gelatin (pHEMA-gelatin), and chitosan-agarose-gelatin in mice. All the cryogels were synthesized at subzero temperature and were implanted subcutaneously in C57Bl/10.Q inbred mice. Both local and systemic toxicities were negligible as determined by serum tumor necrosis factor α analysis and histology of surrounding tissues nearby the implants. Complete integration of cryogels into the surrounding tissues with neovascular formation was evident in all the mice. At the implantation site, massive infiltration of macrophages and few dendritic cells were observed but neutrophils and mast cells were clearly absent. Macrophage infiltrations were observed even inside the pores of cryogel implants. To ascertain whether oxidative radicals are involved in the cryogel degradation, we implanted these gels in mice deficient for reactive oxygen species (ROS) production. Rapid gel degradation was observed in the absence of ROS, and there was no significant difference in the biodegradation of these cryogels between ROS sufficient and deficient mice thereby excluding any major role for ROS in this process. Thus, we demonstrate the biocompatibility and ROS-independent biodegradable properties of cryogels that could be useful for tissue-specific tissue engineering applications.

Characterization of chemically defined poly-N-isopropylacrylamide based copolymeric adjuvants.

PNiPAAm is a thermo-responsive polymer with an adjuvant activity. To identify the minimal chemical structure present within PNiPAAm responsible for its adjuvant property, three different constituent polymers with specific functional groups were synthesized through free radical reaction and tested their adjuvant potential along with PNiPAAm. Among them, polymer with isopropyl attached to an amide showed maximal adjuvant activity in rodents followed by polymer with amide or ketone functional groups. However, secondary amine containing polymer did not show any adjuvant activity. In addition, to improve the adjuvant properties of PNiPAAm, we incorporated an affinity ligand, boronate. At first, we synthesized and characterized the dual responsive copolymers PNiPAAm-co-VPBA and PNiPAAm-co-VPBA-co-DMAEMA. Biocompatibility of these copolymers was confirmed both in vitro and in vivo. Mice injected with these copolymers mixed with collagen (CII) developed significant levels of anti-CII antibodies comprising of all the major IgG subclasses and an increased T cell activation. At the injection site, massive infiltration of immune cells was observed. However, only PNiPAAm-co-VPBA-co-DMAEMA-CII induced arthritis in mice after injection of 0.5M fructose confirming the importance of effective release of CII from the polymer for its adjuvant activity. Thus, a fine balance of hydrophobicity and hydrophilicity promotes adjuvant properties and continuous release of antigen, in this case CII, from polymer is essential for its adjuvant activity.

Neuroendocrine profile in a rat model of psychosocial stress: relation to oxidative stress.

AIMS: Psychosocial stress alters the hypothalamic-pituitary-adrenal axis (HPA-axis). Increasing evidence shows a link between these alterations and oxidant elevation. Oxidative stress is implicated in the stress response and in the pathogenesis of neurologic and psychiatric diseases. NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the central nervous system. Here, we investigated the contributory role of NOX2-derived ROS to the development of neuroendocrine alterations in a rat model of chronic psychosocial stress, the social isolation. RESULTS: Significant elevations in the hypothalamic levels of corticotropin-releasing factor and plasmatic adrenocorticotropic hormone were observed from 4 weeks of social isolation. Increased levels of peripheral markers of the HPA-axis (plasmatic and salivary corticosterone) were observed at a later time point of social isolation (7 weeks). Alteration in the exploratory activity of isolated rats followed the same time course. Increased expression of markers of oxidative stress (8-hydroxy-2-deoxyguanosine [8OhdG] and nitrotyrosine) and NOX2 mRNA was early detectable in the hypothalamus of isolated rats (after 2 weeks), but later (after 7 weeks) in the adrenal gland. A 3-week treatment with the antioxidant/NOX inhibitor apocynin stopped the progression of isolation-induced alterations of the HPA-axis. Rats with a loss-of-function mutation in the NOX2 subunit p47(phox) were totally protected from the alterations of the neuroendocrine profile, behavior, and increased NOX2 mRNA expression induced by social isolation. INNOVATION: We demonstrate that psychosocial stress induces early elevation of NOX2-derived oxidative stress in the hypothalamus and consequent alterations of the HPA-axis, leading ultimately to an altered behavior. CONCLUSION: Pharmacological targeting of NOX2 might be of crucial importance for the treatment of psychosocial stress-induced psychosis.

Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles.

Cell proliferation and long-term production of monoclonal antibody IgG(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 x 9 mm(2)). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine + 2 mM alpha-ketoglutarate or L-alanyl-glutamine (GlutaMAXtrade mark) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine + alpha-ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 x 10(6) cells mL(-1)) and highest specific mAb production rate (3.9 mug mL(-1) 10(-4) viable cell day(-1)), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production.

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices.

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture.

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.