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Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model.

Grimstein C, Choi YK, Wasserfall CH, Satoh M, Atkinson MA, Brantly ML, Campbell-Thompson M, Song S - J Transl Med (2011)

Bottom Line: Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.These results present a new drug for arthritis therapy.Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutics, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA).

Methods: DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA.

Results: Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.

Conclusion: These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.

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Effects of hAAT therapy on T-cells and B-cells. (A) Proliferative response of splenocytes after stimulation with bovine type II collagen (bCII, 10 μg/ml). Splenocytes (4 × 105 cells/well, in 96-well plate) were isolated on day 28 after rAAV8-hAAT injection. Black bar, AAT gene therapy group (n = 6); open bar, control group (n = 4). Data are expressed as the stimulation index, determined by calculating the ratio of cell proliferation with antigen (measured in counts per minute, cpm) relative to that with medium alone (mean+SD). (B) Cytokine production from bCII-stimulated (100 μg/ml) splenocytes. Values are the mean+SD of each group (n = 6 for rAAV8-hAAT group, black bars; n = 4 for saline group, open bars). (C) Serum level of BAFF in hAAT treated mice (black bar, n = 9, day 35) and control mice (open bar, n = 7). Data is expressed as mean+SD. (D) BAFF serum level in rAAV8-hAAT treated mice (black bar, n = 10, day 28) and control (open bar, n = 10). In vitro effect of hAAT on (E) BAFF secretion into culture medium measured by ELISA and (F) BAFF gene expression determined by real-time PCR. Murine macrophages (RAW 264.7) were treated with hAAT (0.5mg/ml, black bar). Culture medium served as control (open bar). Both experiments were performed in quadruplicates and repeated twice. Data is expressed as mean+SD. *p < 0.05, **p < 0.01.
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Figure 6: Effects of hAAT therapy on T-cells and B-cells. (A) Proliferative response of splenocytes after stimulation with bovine type II collagen (bCII, 10 μg/ml). Splenocytes (4 × 105 cells/well, in 96-well plate) were isolated on day 28 after rAAV8-hAAT injection. Black bar, AAT gene therapy group (n = 6); open bar, control group (n = 4). Data are expressed as the stimulation index, determined by calculating the ratio of cell proliferation with antigen (measured in counts per minute, cpm) relative to that with medium alone (mean+SD). (B) Cytokine production from bCII-stimulated (100 μg/ml) splenocytes. Values are the mean+SD of each group (n = 6 for rAAV8-hAAT group, black bars; n = 4 for saline group, open bars). (C) Serum level of BAFF in hAAT treated mice (black bar, n = 9, day 35) and control mice (open bar, n = 7). Data is expressed as mean+SD. (D) BAFF serum level in rAAV8-hAAT treated mice (black bar, n = 10, day 28) and control (open bar, n = 10). In vitro effect of hAAT on (E) BAFF secretion into culture medium measured by ELISA and (F) BAFF gene expression determined by real-time PCR. Murine macrophages (RAW 264.7) were treated with hAAT (0.5mg/ml, black bar). Culture medium served as control (open bar). Both experiments were performed in quadruplicates and repeated twice. Data is expressed as mean+SD. *p < 0.05, **p < 0.01.

Mentions: In order to further elucidate the underlying mechanism of the anti-arthritic effect of hAAT, we performed additional studies focusing on the effect of AAT on T-cell and B-cell activity. Since CIA is a T-cell-mediated autoimmune disease, the effect of hAAT on T-cell function was examined in a T-cell proliferation assay. As shown in Figure 6A, treatment of rAAV8-hAAT did not change the antigen specific T-cell response after isolated splenocytes were restimulated ex vivo with bCII. Similarly, bCII induced cytokine release (IFN-γ, IL-4, IL-10, TNF-α, IL-2) from isolated splenocytes did not show any significant differences between treatment and control group (Figure 6B). The effect of hAAT therapy on B-cell activity was examined by determination of serum levels of B-cell activating factor of the TNF-α family (BAFF), which has emerged as a crucial factor for B-cell expansion and function. Interestingly, both hAAT protein as well as AAV8 mediated hAAT gene therapy resulted in significantly decreased serum levels of BAFF compared to control group (Figure 6C, 6D). Since BAFF is mainly secreted from monocytes and macrophages, we tested the effect of hAAT on BAFF production in vitro. Murine macrophages (RAW264.7) were treated with hAAT. Culture medium served as control. Protein secretion into the culture medium was determined by ELISA and mRNA expression was quantified by real-time PCR. As shown in Figure 6E, BAFF levels in culture medium were significantly lower in the AAT treated group than those in the control group. Similarly, mRNA expression levels of BAFF were also significantly decreased in AAT treated group (Figure 6F). Together these results suggest that the anti-arthritic effect of AAT is in part through the inhibition of B-cell activation.


Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model.

Grimstein C, Choi YK, Wasserfall CH, Satoh M, Atkinson MA, Brantly ML, Campbell-Thompson M, Song S - J Transl Med (2011)

Effects of hAAT therapy on T-cells and B-cells. (A) Proliferative response of splenocytes after stimulation with bovine type II collagen (bCII, 10 μg/ml). Splenocytes (4 × 105 cells/well, in 96-well plate) were isolated on day 28 after rAAV8-hAAT injection. Black bar, AAT gene therapy group (n = 6); open bar, control group (n = 4). Data are expressed as the stimulation index, determined by calculating the ratio of cell proliferation with antigen (measured in counts per minute, cpm) relative to that with medium alone (mean+SD). (B) Cytokine production from bCII-stimulated (100 μg/ml) splenocytes. Values are the mean+SD of each group (n = 6 for rAAV8-hAAT group, black bars; n = 4 for saline group, open bars). (C) Serum level of BAFF in hAAT treated mice (black bar, n = 9, day 35) and control mice (open bar, n = 7). Data is expressed as mean+SD. (D) BAFF serum level in rAAV8-hAAT treated mice (black bar, n = 10, day 28) and control (open bar, n = 10). In vitro effect of hAAT on (E) BAFF secretion into culture medium measured by ELISA and (F) BAFF gene expression determined by real-time PCR. Murine macrophages (RAW 264.7) were treated with hAAT (0.5mg/ml, black bar). Culture medium served as control (open bar). Both experiments were performed in quadruplicates and repeated twice. Data is expressed as mean+SD. *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Effects of hAAT therapy on T-cells and B-cells. (A) Proliferative response of splenocytes after stimulation with bovine type II collagen (bCII, 10 μg/ml). Splenocytes (4 × 105 cells/well, in 96-well plate) were isolated on day 28 after rAAV8-hAAT injection. Black bar, AAT gene therapy group (n = 6); open bar, control group (n = 4). Data are expressed as the stimulation index, determined by calculating the ratio of cell proliferation with antigen (measured in counts per minute, cpm) relative to that with medium alone (mean+SD). (B) Cytokine production from bCII-stimulated (100 μg/ml) splenocytes. Values are the mean+SD of each group (n = 6 for rAAV8-hAAT group, black bars; n = 4 for saline group, open bars). (C) Serum level of BAFF in hAAT treated mice (black bar, n = 9, day 35) and control mice (open bar, n = 7). Data is expressed as mean+SD. (D) BAFF serum level in rAAV8-hAAT treated mice (black bar, n = 10, day 28) and control (open bar, n = 10). In vitro effect of hAAT on (E) BAFF secretion into culture medium measured by ELISA and (F) BAFF gene expression determined by real-time PCR. Murine macrophages (RAW 264.7) were treated with hAAT (0.5mg/ml, black bar). Culture medium served as control (open bar). Both experiments were performed in quadruplicates and repeated twice. Data is expressed as mean+SD. *p < 0.05, **p < 0.01.
Mentions: In order to further elucidate the underlying mechanism of the anti-arthritic effect of hAAT, we performed additional studies focusing on the effect of AAT on T-cell and B-cell activity. Since CIA is a T-cell-mediated autoimmune disease, the effect of hAAT on T-cell function was examined in a T-cell proliferation assay. As shown in Figure 6A, treatment of rAAV8-hAAT did not change the antigen specific T-cell response after isolated splenocytes were restimulated ex vivo with bCII. Similarly, bCII induced cytokine release (IFN-γ, IL-4, IL-10, TNF-α, IL-2) from isolated splenocytes did not show any significant differences between treatment and control group (Figure 6B). The effect of hAAT therapy on B-cell activity was examined by determination of serum levels of B-cell activating factor of the TNF-α family (BAFF), which has emerged as a crucial factor for B-cell expansion and function. Interestingly, both hAAT protein as well as AAV8 mediated hAAT gene therapy resulted in significantly decreased serum levels of BAFF compared to control group (Figure 6C, 6D). Since BAFF is mainly secreted from monocytes and macrophages, we tested the effect of hAAT on BAFF production in vitro. Murine macrophages (RAW264.7) were treated with hAAT. Culture medium served as control. Protein secretion into the culture medium was determined by ELISA and mRNA expression was quantified by real-time PCR. As shown in Figure 6E, BAFF levels in culture medium were significantly lower in the AAT treated group than those in the control group. Similarly, mRNA expression levels of BAFF were also significantly decreased in AAT treated group (Figure 6F). Together these results suggest that the anti-arthritic effect of AAT is in part through the inhibition of B-cell activation.

Bottom Line: Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.These results present a new drug for arthritis therapy.Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutics, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA).

Methods: DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA.

Results: Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.

Conclusion: These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.

Show MeSH
Related in: MedlinePlus