Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies.

CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

γδ T cells can either enhance or inhibit an adaptive immune response, but the mechanisms involved are not fully understood. Given that CD73 is the main enzyme responsible for conversion of AMP into the immunosuppressive molecule adenosine, we investigated its role in the regulatory function of γδ T cells in experimental autoimmune uveitis (EAU). We found that γδ T cells expressed different amounts of CD73 during the different stages of EAU and that low CD73 expression on γδ T cells correlated with enhanced Th17 response-promoting activity. Functional comparison of CD73-deficient and wild-type B6 (CD73+/+) mice showed that failure to express CD73 decreased both the enhancing and suppressive effects of γδ T cells on EAU. We also demonstrated that γδ T cells expressed different amounts of CD73 when activated by different pathways, which enabled them to either enhance or inhibit an adaptive immune response. Our results demonstrate that targeting CD73 expression on γδ T cells may allow us to manipulate their pro- or anti-inflammatory effect on Th17 responses.

An A2B Adenosine Receptor Agonist Promotes Th17 Autoimmune Responses in Experimental Autoimmune Uveitis (EAU) via Dendritic Cell Activation.

We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs). In this study, we further demonstrate that treatment of C57BL/6 (B6) mice with a selective A2B adenosine receptor (A2BR) agonist greatly enhanced the development of experimental autoimmune uveitis (EAU), whereas treatment with an A2BR antagonist significantly ameliorated severity of EAU. The A2BR agonist-treated mice showed augmented Th17, but not Th1, responses. Mechanistic studies showed that the A2BR agonist-induced enhancement of the Th17 response was significantly lower when TCR-δ-/- mice received the same treatment and that transfer of γδ T cells into TCR-δ-/- mice partially restored this effect. We also showed that dendritic cells (DCs) from A2BR agonist-treated mice showed a significantly increased ability to activate γδ T cells and Th17 autoreactive T cells. Thus, our previous studies have shown that, in EAU, activated γδ T cells possess greatly increased ability to enhance Th17 autoimmune responses. In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development. Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c(+)Gr-1(+) dendritic cell subset that promotes the Th17 response.

Adenosine is one of the major molecules associated with inflammation. We have previously reported that an adenosine receptor (AR) agonist has an enhancing effect on Th17 autoimmune responses, even though it suppressed Th1 responses. To determine the mechanism involved, we have examined the effect of AR agonists on mouse bone marrow dendritic cell (BMDC) differentiation and function. We show that mouse bone marrow cells (BMCs) differentiated into CD11c(+)Gr-1(+) dentritic cells (DCs) when cultured in granulocyte macrophage colony-stimulating factor (GM-CSF)-containing medium containing an AR agonist. The non-selective AR agonist NECA and an A2BR-specific agonist had a similar effect, and the effect of NECA could be blocked by an A2BR-specific antagonist. Unlike CD11c(+)Gr-1(-) BMDCs, which have a greater stimulatory effect on Th1 T cells than Th17 cells, CD11c(+)Gr-1(+) BMDCs had a greater stimulatory effect on Th17 autoreactive T cells than on Th1 autoreactive T cells and this effect depended on γδ T cell activation.

Anti-inflammatory or proinflammatory effect of an adenosine receptor agonist on the Th17 autoimmune response is inflammatory environment-dependent.

Adenosine is a key endogenous signaling molecule that regulates a wide range of physiological functions, including immune system function and inflammation. Studies have shown that adenosine receptor (AR) agonists can be either anti-inflammatory or proinflammatory in immune responses and in inflammation, and the clarification of the mechanisms causing these opposing effects should provide a better guide for therapeutic intervention. Whereas previous studies mostly examined the effects of AR agonists on Th1-type immune responses, in this study, we compared their effect on Th17 and Th1 autoimmune responses in experimental autoimmune uveitis, a mouse model of human uveitis induced by immunization with the human interphotoreceptor retinoid-binding protein peptides 1-20. We showed that injection of mice with a nonselective AR agonist, 5'-N-ethylcarboxamidoadenosine (NECA), at an early stage after immunization had an inhibitory effect on both Th1 and Th17 responses, whereas injection of the same amount of NECA at a late stage inhibited the Th1 response but had an enhancing effect on the Th17 response. We also showed that the effects of NECA on Th1 and Th17 responses were completely dissociated, that the enhancing effect of NECA on Th17 responses was modulated by γδ T cells, and that the response of γδ T cells to NECA was determined by their activation status. We conclude that the inflammatory environment has a strong impact on converting the effect of AR agonist on the Th17 autoimmune response from anti-inflammatory to proinflammatory. Our observation should help in the designing of better AR-targeted therapies.

Roles of the adenosine receptor and CD73 in the regulatory effect of γδ T cells.

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αß T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses.

Subretinal transfection of chitosan-loaded TLR3-siRNA for the treatment of experimental autoimmune uveitis.

The local interaction between retinal pigment epithelium (RPE) and immigrated effector T cells is crucial for the pathogenesis of autoimmune uveitis. After being activated by the pattern recognition receptors (PRRs) signaling pathway, RPE can present the antigen reactivated invading autoreactive T cells, resulting in uveitis. In the present study, we showed that the transfection of chitosan-loaded TLR3-siRNA toward RPE could effectively remit experimental autoimmune uveitis (EAU) in B10RIII mice. Initially, we verified the constitutive expression of Tlr3 in RPE at high levels, which was not altered in response to TNFα, IFNγ and IL-17A treatments. Compared with other TLRs, the activation of TLR3 signaling following polyIC treatment resulted in increased IL-6 and IFNγ secretion from and MHCII expression on RPE. It is polyIC-, but not other TLR ligands, treated RPE showed significant synergetic effect with IL-17 on stimulating RPE secreting CXCL8 and CCl2, which might be resulted from elevated Il17ra expression in RPE following polyIC treatment. Furthermore, polyIC-treated RPE caused a robust stimulation of differentiation of CD4 cell toward Th1 or Th17 cells, in addition to the secretion of the cytokines IFNγ and IL-17. The in vitro knockdown of TLR3 expression in RPE by chitosan/TLR3-siRNA transfection could effectively block polyIC-induced MHCII expression, pro-inflammatory cytokine secretion and autoreactive CD4 cell activation. Studies conducted in firefly luciferase gene transgenic mice demonstrated that the subretinal CS/Luci-siRNA transfection specifically reduced the luciferase activity in RPE but not in the liver and spleen. Finally, the CS/TLR3-siRNA was locally administered in the EAU induced B10RIII mice. The results revealed that chitosan-mediated TLR3-siRNA transfection had a significant therapeutic effect on either delaying the outbreak or remitting the severity of uveitis.

Retinoic acid inhibits CD25+ dendritic cell expansion and γδ T-cell activation in experimental autoimmune uveitis.

PURPOSE: We determined the mechanism by which all-trans retinoic acid (ATRA) inhibits experimental autoimmune uveitis (EAU) and determined the role of γδ T cells in this autoimmune disease. METHODS: C57BL/6 (B6) mice were immunized with the uveitogenic, interphotoreceptor retinoid-binding protein1-20 peptide (IRBP1-20) in complete Freund's adjuvant (CFA), with or without a preceding ATRA treatment. Responses and pathogenic activity of Th1- and Th17-autoreactive T cells were compared, and the effects of ATRA on γδ T cells and CD25(+) dendritic cell (DC) subset were determined. Interactions among uveitogenic T cells, DC subsets, and γδ T cells were investigated. RESULTS: Administration of ATRA to B6 mice in which EAU was induced suppressed the response of Th17 autoreactive T cells, which was associated with decreased generation of the CD25(+) DC subset and suppressed activation of γδ T cells. Adoptively transferred γδ T cells isolated from ATRA-treated mice showed a diminished ability to promote the activation of Th17 autoreactive T cells in vitro and in vivo compared to γδ T cells from untreated donors. CONCLUSIONS: ATRA inhibits the expansion of CD25(+) DCs and γδ T-cell activation, thereby restraining the Th17 autoreactive T-cell response.

Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing.

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. However, its application was greatly hampered by the rapid degradation and poor cellular uptake. Recently, chitosan (CS) and its derivant have been considered as a promising siRNA transporter with the advantages of low toxicity, good biodegradability and biocompatibility. Chitosan of different molecular weight (Mw) and degrees of deacetylation (DD) showed significantly varied target gene silencing efficacy, and it is still not well clarified how these characteristics influence CS mediated siRNA transfection. To compare the aspects of cell permeability and intracellular unpacking of CS/siRNA complex on the effect of CS/siRNA transfection. A radiolabeled siRNA, targeting firefly luciferase gene, was loaded by chitosan of different molecular weight (varying from 2000 to 800,000 Da) and subjected to the transfection against MDA-MB-231/Luc human breast cancer cell line which stably expressed knocked in firefly Luciferase reporter gene. Following transfection intracellular radioactivity was measured to represent cell entrance ability of the CS/siRNA, while, luciferase activity in the cell lysate was also determined to reflect target gene silencing effect. The results revealed that although low molecular weight chitosan (LMWC) condensed siRNA has the highest cell permeability of almost two folds of medium molecular weight chitosan and lipofactamine, its target gene silencing effect is really low of almost eight times less than lipofectamine. This conspicuous contradiction gave us the hypothesis that LMWC generated more condensed CS/siRNA complex to facilitate cell entrance but the tight electrostatic interaction probably limited intracellular siRNA unpacking as well and unfavorably hindered target gene silencing as the final consequence. To approve this hypothesis a phosphorylatable short peptide conjugated LMWC was adopt to promote intracellular siRNA unpacking. Which was demonstrated of perfect target gene knock down ability to the extent of being even superior to lipofactamine 2000. In a conclusion, low molecular weight chitosan has the great potential to be an ideal siRNA vehicle if the issue of siRNA unpacking could be properly resolved.

Optimize nuclear localization and intra-nucleus disassociation of the exogene for facilitating transfection efficacy of the chitosan.

Previously, we had reported improving transfection efficiency of the chitosan-plasmid DNA (CS/pDNA)complex via enhancing intracellular unpacking of the exogene by the utilization of phosphorylatable short peptide conjugated chitosan (pSP-CS). In this article, we addressed a novel strategy of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) modification for further optimization of the transfection efficiency. NNS, consisting of "PKKRKVREEAIKFSEEQRFRR", contained a SV40 nucleus localization signal and a potentially phosphorylatable serine residue. The short peptide could be selectively phosphorylated in the nucleus in various mammalian cells. This phosphorylatable NNS (pNNS) was conjugated to chitosan and combined with Cy3 fluorescence labeled plasmid DNA to form a pNNS-CS/pDNA complex. In vitro phosphorylation and DNA releasing assays verified that pNNS could be effectively and selectively phosphorylated by nucleic lysate, hence promoting pDNA unpacking from the complex. Thereafter, C2C12 myoblast cells were transfected. Nuclear localization of the pDNA was represented by the fluorescence in the nucleus and transfection efficiency was determined by the expression of the luciferase reporter gene, which is carried by the plasmid DNA. The results revealed that, compared with lipofactamine2000 and the previously reported pSP-CS, pNNS-CS could transport more pDNA into the nucleus and intensively augment luciferase reporter gene expression. In conclusion, nucleus localization and unpacking from the delivery vector are both critical factors in influencing exogene expression, and pNNS modification is valuable in improving transfection efficacy of the chitosan.

Phosphorylatable short peptide conjugation for facilitating transfection efficacy of CS/DNA complex.

Previously, we had demonstrated that enhancing intracellular unpacking of exogene from its chitosan carrier by promoting chitosan degradation could markedly improve transfection efficiency of the CS/DNA complex. In this article we addressed a novel strategy of phosphorylatable short peptide modification for further facilitating intracellular DNA unpacking and optimizing transfection efficiency of the CS/DNA complex. A short peptide (SP) with the amino acid composition of "LLLRRRDNEY*FY*VRRLL" containing two potentially phosphorylatable tyrosine residues was synthesized. The short peptide could be phosphorylated by constitutively expressed cytoplasmic protein kinase Jak2. The SP was conjugated to chitosan and combined with GFP/luciferase reporter gene plasmid DNA to form SP-CS/DNA complex. In vitro phosphorylation and DNA releasing assays verified that mammalian cell lysate could effectively phosphorylate SP and hence promote plasmid DNA unpacking from the SP-CS carrier. Thereafter, C2C12 myoblast cells were transfected by SP-CS/DNA and the transfection efficiency was presented by the expression of GFP and luciferase reporters. Further more, multiple cell lines were transfected by SP-CS/DNA complexes loading luciferase reporter gene. Results revealed that, compared with CS, SP-CS could intensively augment the transfection efficiency to the level of near lipofectamine 2000.

Improved transfection efficiency of CS/DNA complex by co-transfected chitosanase gene.

Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatuscsn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by beta-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.

[Interleukin-6 and tumor necrosis factor-alpha levels of endothelial cells in different hypoxia modes: in vitro experiment].

OBJECTIVE: To investigate the damage of different patterns of intermittent hypoxia (IH) and continuous hypoxia (CH) on endothelial cells. METHODS: Human umbilical vein endothelial cells of the line ECV304 were cultured in a program-controlled gas delivery system newly developed and divided into 8 groups to undergo different IH/reoxygenation (ROX) cycles so as to simulate the patterns of hypoxic episode seen in recurrent apnea and chronic obstructive pulmonary disease: intermittent normoxia (IN) group (exposed to 21% O2 15 s/21% O2 225 s for 60 cycles), IH group (exposed to 1.5% O2 15 s/21% O2 225 s for 30 or 60 cycles), IH hypercapnia group (exposed to 1.5% O2 and 20% CO2 15 s/21% O2 and 5% CO2 225 s, for 60 cycles), continuous hypoxia (CH group, exposed to 1.5% or 10% O2 for 15, 30 or 60 min), CH hypercapnia group (exposed to 10% O2 and 10% CO2 for 15, 30 or 60 min), CH added to IH group (exposed to 1.5% O2 15 s/10% O2 225 s for 60 cycles), different intermittent hypoxia extent group (exposed to 1.5% or 10% O2 15 s/21% O2 225 s for 60 cycles), different intermittent hypoxia frequency group (exposed to 1.5% O2 15 s/21% O2 225 s 315 s, 495 s or 105 s for 60 cycles), and different intermittent hypoxia duration group (exposed to 1.5% O2 15 s or 30 s/21% O2 225 s for 60 cycles). Then ELISA was conducted to examine the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha) Bicinchoninic acid method was used to standardize the cell total protein level. RESULTS: The levels of IL-6 and TNFalpha levels in the IH group were (770.40 +/- 21.60) and (126.93 +/- 2.58) pg.ml(-1).100 mg protein(-1) respectively, both significantly higher than those in the IN group [(374.06 +/- 38.10) and (31.96 +/- 13.64) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002], but significantly lower than those in the IH hypercapnia group [(829.27 +/- 7.16) and (78.77 +/- 4.00) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002]. The IL-6 levels of the CH hypercapnia 15, 30, and 60 min subgroups were all significantly higher than those of the corresponding CH subgroup (U = 0.000, P = 0.002). The IL-6 and TNFalpha levels of the CH added to IH group were (536.74 +/- 14.97) and (51.10 +/- 6.80) pg.ml(-1).100 mg protein(-1) respectively, both were significantly higher than those of the IN group, but significantly lower than those of the IH group (chi(2) = 23.4, P < 0.05). The levels of IL-6 and TNFalpha increased along with the increase of the IH degree (chi(2) = 23.4, P < 0.05). The level changes of IL-6 and TNFalpha of the groups with different intermittent hypoxia frequency and with different intermittent hypoxia duration were complicated. CONCLUSION: IH and CH significantly damage the endothelial cells dose-dependently, especially combined with hypercapnia. In IH/ROX, the inflammatory damage comes from ROX phase but not IH phase. Hypoxia duration and hypoxia frequency are also important parameters in the activation of inflammation.

[In vitro osteogenesis of the compound of chitosan and recombinant human bone morphogenetic protein 2].

OBJECTIVE: To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). METHODS: Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freeze-drying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at the density of 2 x 10(4)/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semi-quantitative RT-PCR with housekeeping gene beta-tubulin as internal-standard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected to the detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition and was treated with the same as group A. Besides these 2 groups, another 2 rhBMP-2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. RESULTS: SEM showed that cells attached closely to the complex and grew well. In group A, the expression level (1.28+/-0.17) of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56+/-0.09) of the control group A, being statistically significantly different (P< 0. 05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. CONCLUSION: The complex made of rhBMP-2 and chitosan-gelatin scaffolds has strong osteogenesis ability in vitro.