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RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis.

Zheng X, Suzuki M, Zhang X, Ichim TE, Zhu F, Ling H, Shunnar A, Wang MH, Garcia B, Inman RD, Min WP - Arthritis Res. Ther. (2010)

Bottom Line: Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Surgery, Pathology, Microbiology and Immunology, University of Western Ontario, 1393 Western Road, London, Ontario, N6G 1G9, Canada. xzheng26@uwo.ca

ABSTRACT

Introduction: We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods: Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results: Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions: These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.

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Related in: MedlinePlus

Knockdown of CD40 reduces allogenicity in DCs. DBA-derived DCs were cultured as described in Materials and methods. DCs were transfected with CD40 siRNA using Genesilencer. Forty-eight hours after gene silencing, reduced gene expression of CD40 was detected by RT-PCR a) and flow cytometry b), respectively, without affecting CD80 gene expression determined by flow cytometry c). CD40-silenced DCs were also used to co-culture allogeneic (BALB/c) T cells. T cell proliferation was assessed in MLR d). Data are presented as mean ± SEM. Results represent one of three experiments. * = P < 0.05 versus control siRNA.
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Figure 1: Knockdown of CD40 reduces allogenicity in DCs. DBA-derived DCs were cultured as described in Materials and methods. DCs were transfected with CD40 siRNA using Genesilencer. Forty-eight hours after gene silencing, reduced gene expression of CD40 was detected by RT-PCR a) and flow cytometry b), respectively, without affecting CD80 gene expression determined by flow cytometry c). CD40-silenced DCs were also used to co-culture allogeneic (BALB/c) T cells. T cell proliferation was assessed in MLR d). Data are presented as mean ± SEM. Results represent one of three experiments. * = P < 0.05 versus control siRNA.

Mentions: Immature DCs are characterized by relatively low expression of stimulatory signals and higher levels of inhibitory signals to T cells [13]. Previously we demonstrated feasibility of manipulating such signals using siRNA on DCs in order to generate immunomodulatory DCs in a reproducible manner by silencing IL-12p35 [14]. We have postulated that specific gene knock-down may be a more attractive and defined means of immune modulation as opposed to chemically generated immature DCs, which we have also demonstrated to inhibit disease progression in CIA [15]. Here we sought to develop inhibitory DCs through gene silencing of CD40. DCs were generated from bone marrow progenitors by standard IL-4 and GM-CSF culture and transfected on Day 6 with CD40-siRNA. Specific knockdown of CD40 mRNA and protein was seen in DCs by RT-PCR (Figure 1A), and flow cytometry (Figure 1B), respectively, as compared to DCs transfected with control scrambled siRNA. Silencing was specific in that CD 40 siRNA did not change gene expression of CD80 in DCs (Figure 1C).


RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis.

Zheng X, Suzuki M, Zhang X, Ichim TE, Zhu F, Ling H, Shunnar A, Wang MH, Garcia B, Inman RD, Min WP - Arthritis Res. Ther. (2010)

Knockdown of CD40 reduces allogenicity in DCs. DBA-derived DCs were cultured as described in Materials and methods. DCs were transfected with CD40 siRNA using Genesilencer. Forty-eight hours after gene silencing, reduced gene expression of CD40 was detected by RT-PCR a) and flow cytometry b), respectively, without affecting CD80 gene expression determined by flow cytometry c). CD40-silenced DCs were also used to co-culture allogeneic (BALB/c) T cells. T cell proliferation was assessed in MLR d). Data are presented as mean ± SEM. Results represent one of three experiments. * = P < 0.05 versus control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875641&req=5

Figure 1: Knockdown of CD40 reduces allogenicity in DCs. DBA-derived DCs were cultured as described in Materials and methods. DCs were transfected with CD40 siRNA using Genesilencer. Forty-eight hours after gene silencing, reduced gene expression of CD40 was detected by RT-PCR a) and flow cytometry b), respectively, without affecting CD80 gene expression determined by flow cytometry c). CD40-silenced DCs were also used to co-culture allogeneic (BALB/c) T cells. T cell proliferation was assessed in MLR d). Data are presented as mean ± SEM. Results represent one of three experiments. * = P < 0.05 versus control siRNA.
Mentions: Immature DCs are characterized by relatively low expression of stimulatory signals and higher levels of inhibitory signals to T cells [13]. Previously we demonstrated feasibility of manipulating such signals using siRNA on DCs in order to generate immunomodulatory DCs in a reproducible manner by silencing IL-12p35 [14]. We have postulated that specific gene knock-down may be a more attractive and defined means of immune modulation as opposed to chemically generated immature DCs, which we have also demonstrated to inhibit disease progression in CIA [15]. Here we sought to develop inhibitory DCs through gene silencing of CD40. DCs were generated from bone marrow progenitors by standard IL-4 and GM-CSF culture and transfected on Day 6 with CD40-siRNA. Specific knockdown of CD40 mRNA and protein was seen in DCs by RT-PCR (Figure 1A), and flow cytometry (Figure 1B), respectively, as compared to DCs transfected with control scrambled siRNA. Silencing was specific in that CD 40 siRNA did not change gene expression of CD80 in DCs (Figure 1C).

Bottom Line: Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Surgery, Pathology, Microbiology and Immunology, University of Western Ontario, 1393 Western Road, London, Ontario, N6G 1G9, Canada. xzheng26@uwo.ca

ABSTRACT

Introduction: We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods: Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results: Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions: These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.

Show MeSH
Related in: MedlinePlus