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IL-28 supplants requirement for T(reg) cells in protein sigma1-mediated protection against murine experimental autoimmune encephalomyelitis (EAE).

Rynda A, Maddaloni M, Ochoa-Repáraz J, Callis G, Pascual DW - PLoS ONE (2010)

Bottom Line: Conventional methods to induce tolerance in humans have met with limited success.To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP(130-151)) was genetically fused to OVA to psigma1 (PLP:OVA-psigma1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10(+) forkhead box P3 (FoxP3)(+) CD25(+)CD4(+) T(reg) and IL-4(+)CD25(-)CD4(+) Th2 cells.Thus, psigma1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America.

ABSTRACT
Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (psigma1), may circumvent these shortcomings based upon the recent finding that when reovirus psigma1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP(130-151)) was genetically fused to OVA to psigma1 (PLP:OVA-psigma1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10(+) forkhead box P3 (FoxP3)(+) CD25(+)CD4(+) T(reg) and IL-4(+)CD25(-)CD4(+) Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-psigma1, and adoptive transfer of Ag-specific T(reg) or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-psigma1's protective capacity, triggering TGF-beta-mediated inflammation; however, concomitant inactivation of TGF-beta and CD25 reestablished PLP:OVA-psigma1-mediated protection by IL-28-producing FoxP3(+)CD25(-)CD4(+) T cells. Thus, psigma1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.

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PLP:OVA-pσ1 induces IL-10-producing Treg and IL-4-producing FoxP3+Th2 cells.Mice were dosed with 100 µg of PLP:OVA-pσ1 A and C or with PBS B and C fourteen days before challenge with PLP139–151 peptide and sacrificed 2 weeks later. A and B. Lymphocytes from head and neck LNs (HNLNs) and spleens were isolated, cultured with 30 µg/ml of PLP139–151 for 3 days, and stained for expression of extracellular (CD25, CD4 and TGF-β), and intracellular markers (FoxP3, IL-10, and IL-4). Presented FACS plots show cells isolated from spleens with respective isotype controls provided in inserts. Unlike PBS-dosed mice, PLP:OVA-pσ1 administration induced significant enrichment in FoxP3+ Treg cells and CD25− Th2 cells in mice. In contrast to PBS-derived CD4+ T cells, FoxP3+ Treg cells from PLP:OVA-pσ1-dosed mice produced predominantly IL-10, whereas PLP:OVA-pσ1-derived FoxP3+ Th2 cells produced IL-4. Average percentage of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS. C. CD25+CD4+ and CD25−CD4+ T cells were bead-sorted from combined HNLN, MLN, and splenic lymphocytes and in vitro stimulated with plate bound anti-CD3 and soluble anti-CD28 mAbs for 72 h. Collected supernatants were analyzed by cytokine ELISA. Negligible amounts of proinflammatory cytokines were secreted by CD4+ T cells isolated from PLP:OVA-pσ1-dosed and challenged mice. Mean ± SEM of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS.
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pone-0008720-g002: PLP:OVA-pσ1 induces IL-10-producing Treg and IL-4-producing FoxP3+Th2 cells.Mice were dosed with 100 µg of PLP:OVA-pσ1 A and C or with PBS B and C fourteen days before challenge with PLP139–151 peptide and sacrificed 2 weeks later. A and B. Lymphocytes from head and neck LNs (HNLNs) and spleens were isolated, cultured with 30 µg/ml of PLP139–151 for 3 days, and stained for expression of extracellular (CD25, CD4 and TGF-β), and intracellular markers (FoxP3, IL-10, and IL-4). Presented FACS plots show cells isolated from spleens with respective isotype controls provided in inserts. Unlike PBS-dosed mice, PLP:OVA-pσ1 administration induced significant enrichment in FoxP3+ Treg cells and CD25− Th2 cells in mice. In contrast to PBS-derived CD4+ T cells, FoxP3+ Treg cells from PLP:OVA-pσ1-dosed mice produced predominantly IL-10, whereas PLP:OVA-pσ1-derived FoxP3+ Th2 cells produced IL-4. Average percentage of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS. C. CD25+CD4+ and CD25−CD4+ T cells were bead-sorted from combined HNLN, MLN, and splenic lymphocytes and in vitro stimulated with plate bound anti-CD3 and soluble anti-CD28 mAbs for 72 h. Collected supernatants were analyzed by cytokine ELISA. Negligible amounts of proinflammatory cytokines were secreted by CD4+ T cells isolated from PLP:OVA-pσ1-dosed and challenged mice. Mean ± SEM of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS.

Mentions: CD4+ T cells isolated from PLP:OVA-pσ1- and PBS-dosed mice after EAE challenge were evaluated by flow cytometry to determine their Treg cell composition. Nasal PLP:OVA-pσ1-dosed mice showed a significant augmentation in FoxP3+ CD25+CD4+ T cells and FoxP3+ CD25−CD4+ Th2 cells, when compared to PBS-dosed mice (Figure 2). PLP:OVA-pσ1 induced >25% of CD4+CD25+ T cells, of which >93% were FoxP3+ and CD25−CD4+ T cells of which >18% were FoxP3+ (Figure 2A; Table S1), unlike PBS-dosed mice showing only a slight enrichment in Treg cells (∼10%) and CD25− Th2 cells as naive mice (Figure 2B). CD4+ T cells from PLP:OVA-pσ1-dosed mice showed >85% Treg cells expressing IL-10 and nearly as many CD25− Th2 cells producing IL-4 and significantly more TGF-β than those from PBS-dosed mice. Proinflammatory cytokines, IL-17, IL-21, and IFN-γ, were produced primarily by PBS-derived CD4+ T cells and only nominally by those from PLP:OVA-pσ1-dosed mice (Figure 2C). Thus, nasal administration of PLP:OVA-pσ1 induced Ag-specific, anti-inflammatory Treg and Th2 cells.


IL-28 supplants requirement for T(reg) cells in protein sigma1-mediated protection against murine experimental autoimmune encephalomyelitis (EAE).

Rynda A, Maddaloni M, Ochoa-Repáraz J, Callis G, Pascual DW - PLoS ONE (2010)

PLP:OVA-pσ1 induces IL-10-producing Treg and IL-4-producing FoxP3+Th2 cells.Mice were dosed with 100 µg of PLP:OVA-pσ1 A and C or with PBS B and C fourteen days before challenge with PLP139–151 peptide and sacrificed 2 weeks later. A and B. Lymphocytes from head and neck LNs (HNLNs) and spleens were isolated, cultured with 30 µg/ml of PLP139–151 for 3 days, and stained for expression of extracellular (CD25, CD4 and TGF-β), and intracellular markers (FoxP3, IL-10, and IL-4). Presented FACS plots show cells isolated from spleens with respective isotype controls provided in inserts. Unlike PBS-dosed mice, PLP:OVA-pσ1 administration induced significant enrichment in FoxP3+ Treg cells and CD25− Th2 cells in mice. In contrast to PBS-derived CD4+ T cells, FoxP3+ Treg cells from PLP:OVA-pσ1-dosed mice produced predominantly IL-10, whereas PLP:OVA-pσ1-derived FoxP3+ Th2 cells produced IL-4. Average percentage of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS. C. CD25+CD4+ and CD25−CD4+ T cells were bead-sorted from combined HNLN, MLN, and splenic lymphocytes and in vitro stimulated with plate bound anti-CD3 and soluble anti-CD28 mAbs for 72 h. Collected supernatants were analyzed by cytokine ELISA. Negligible amounts of proinflammatory cytokines were secreted by CD4+ T cells isolated from PLP:OVA-pσ1-dosed and challenged mice. Mean ± SEM of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806841&req=5

pone-0008720-g002: PLP:OVA-pσ1 induces IL-10-producing Treg and IL-4-producing FoxP3+Th2 cells.Mice were dosed with 100 µg of PLP:OVA-pσ1 A and C or with PBS B and C fourteen days before challenge with PLP139–151 peptide and sacrificed 2 weeks later. A and B. Lymphocytes from head and neck LNs (HNLNs) and spleens were isolated, cultured with 30 µg/ml of PLP139–151 for 3 days, and stained for expression of extracellular (CD25, CD4 and TGF-β), and intracellular markers (FoxP3, IL-10, and IL-4). Presented FACS plots show cells isolated from spleens with respective isotype controls provided in inserts. Unlike PBS-dosed mice, PLP:OVA-pσ1 administration induced significant enrichment in FoxP3+ Treg cells and CD25− Th2 cells in mice. In contrast to PBS-derived CD4+ T cells, FoxP3+ Treg cells from PLP:OVA-pσ1-dosed mice produced predominantly IL-10, whereas PLP:OVA-pσ1-derived FoxP3+ Th2 cells produced IL-4. Average percentage of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS. C. CD25+CD4+ and CD25−CD4+ T cells were bead-sorted from combined HNLN, MLN, and splenic lymphocytes and in vitro stimulated with plate bound anti-CD3 and soluble anti-CD28 mAbs for 72 h. Collected supernatants were analyzed by cytokine ELISA. Negligible amounts of proinflammatory cytokines were secreted by CD4+ T cells isolated from PLP:OVA-pσ1-dosed and challenged mice. Mean ± SEM of 10 mice/group is depicted. * P<0.05 for PLP:OVA-pσ1 vs. PBS.
Mentions: CD4+ T cells isolated from PLP:OVA-pσ1- and PBS-dosed mice after EAE challenge were evaluated by flow cytometry to determine their Treg cell composition. Nasal PLP:OVA-pσ1-dosed mice showed a significant augmentation in FoxP3+ CD25+CD4+ T cells and FoxP3+ CD25−CD4+ Th2 cells, when compared to PBS-dosed mice (Figure 2). PLP:OVA-pσ1 induced >25% of CD4+CD25+ T cells, of which >93% were FoxP3+ and CD25−CD4+ T cells of which >18% were FoxP3+ (Figure 2A; Table S1), unlike PBS-dosed mice showing only a slight enrichment in Treg cells (∼10%) and CD25− Th2 cells as naive mice (Figure 2B). CD4+ T cells from PLP:OVA-pσ1-dosed mice showed >85% Treg cells expressing IL-10 and nearly as many CD25− Th2 cells producing IL-4 and significantly more TGF-β than those from PBS-dosed mice. Proinflammatory cytokines, IL-17, IL-21, and IFN-γ, were produced primarily by PBS-derived CD4+ T cells and only nominally by those from PLP:OVA-pσ1-dosed mice (Figure 2C). Thus, nasal administration of PLP:OVA-pσ1 induced Ag-specific, anti-inflammatory Treg and Th2 cells.

Bottom Line: Conventional methods to induce tolerance in humans have met with limited success.To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP(130-151)) was genetically fused to OVA to psigma1 (PLP:OVA-psigma1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10(+) forkhead box P3 (FoxP3)(+) CD25(+)CD4(+) T(reg) and IL-4(+)CD25(-)CD4(+) Th2 cells.Thus, psigma1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America.

ABSTRACT
Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (psigma1), may circumvent these shortcomings based upon the recent finding that when reovirus psigma1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP(130-151)) was genetically fused to OVA to psigma1 (PLP:OVA-psigma1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10(+) forkhead box P3 (FoxP3)(+) CD25(+)CD4(+) T(reg) and IL-4(+)CD25(-)CD4(+) Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-psigma1, and adoptive transfer of Ag-specific T(reg) or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-psigma1's protective capacity, triggering TGF-beta-mediated inflammation; however, concomitant inactivation of TGF-beta and CD25 reestablished PLP:OVA-psigma1-mediated protection by IL-28-producing FoxP3(+)CD25(-)CD4(+) T cells. Thus, psigma1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.

Show MeSH
Related in: MedlinePlus