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Deletion of IFT20 in early stage T lymphocyte differentiation inhibits the development of collagen-induced arthritis.

Yuan X, Garrett-Sinha LA, Sarkar D, Yang S - Bone Res (2014)

Bottom Line: Recently, IFT20 was found to be also expressed in non-ciliated T cells and have functions in immune synapse formation and signaling in vitro.However, how IFT20 regulates T-cell development and activation in vivo is still unknown.These results demonstrate that deletion of IFT20 in the early stage of T-cell development inhibited CIA development through regulating T-cell development and the expression of critical cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, School of Dental Medicine, University at Buffalo, The State University of New York , Buffalo, NY 14214, USA.

ABSTRACT
IFT20 is the smallest member of the intraflagellar transport protein (IFT) complex B. It is involved in cilia formation. Studies of IFT20 have been confined to ciliated cells. Recently, IFT20 was found to be also expressed in non-ciliated T cells and have functions in immune synapse formation and signaling in vitro. However, how IFT20 regulates T-cell development and activation in vivo is still unknown. We deleted the IFT20 gene in early and later stages of T-cell development by crossing IFT20(flox/flox) (IFT20(f/f) ) mice with Lck-Cre and CD4-Cre transgenic mice, and investigated the role of IFT20 in T-cell maturation and in the development of T cell-mediated collagen-induced arthritis (CIA). We found that both Lck-Cre/IFT20(f/f) and CD4-Cre/IFT20(f/f) mice were indistinguishable from their wild-type littermates in body size, as well as in the morphology and weight of the spleen and thymus. However, the number of CD4- and CD8-positive cells was significantly lower in thymus and spleen in Lck-Cre/IFT20(f/f) mice. Meanwhile, the incidence and severity of CIA symptoms were significantly decreased, and inflammation in the paw was significantly inhibited in Lck-Cre/IFT20(f/f) mice compared to Lck-Cre/IFT20(+/+) littermates. Deletion IFT20 in more mature T cells of CD4-Cre/IFT20(f/f) mice had only mild effects on the development of T cells and CIA. The expression of IL-1β, IL-6 and TGF-β1 were significantly downregulated in the paw of Lck-Cre/IFT20(f/f) mice, but just slight decreased in CD4-Cre/IFT20(f/f) mice. These results demonstrate that deletion of IFT20 in the early stage of T-cell development inhibited CIA development through regulating T-cell development and the expression of critical cytokines.

No MeSH data available.


Related in: MedlinePlus

Deletion IFT20 in double-negative thymocytes affected the development of CIA. (a) Timeline for the CIA mouse model. Seven- to eight-week-old mice were immunized with 100 μL emulsion containing equal volume of CFA (including 5 mg·mL−1 of M. tuberculosis) and chicken type II collagen solution (2 mg·mL−1 in 0.05 mol·mL−1 acetic acid). The booster injection (Insert site: 3 cm from the base of the tail Injection site: 1.5 cm from the base) was made with the same emulsions 21 days after initial injection (Insert site: 2 cm from the base of the tail Injection site: 0.5 cm from the base). Arthritis usually can be observed 4–6 weeks after the first immunization with the maximum incidence of arthritis at 6–7 weeks. All the experimental mice were harvested at day 66 for the histological analysis. (b) Mean clinical scores±s.d. of arthritis in Lck-Cre/IFT20+/+ (n=6) and Lck-Cre/IFT20f/f mice (n=4) with arthritis (P<0.01 after day 40). (c) Percentage of mice of Lck-Cre/IFT20+/+ and Lck-Cre/IFT20f/f mice that developed arthritis (n=10, P<0.05 after day 40). (d) The maximum clinical score was recorded until day 66 (n=10, *P<0.01). (e) Hind paws of type II collagen immunized Lck-Cre/IFT20+/+ and Lck-Cre/ IFT20f/f mice. (f) The hind paw thickness was measured at day 66 to indicate arthritis development (n=10, *P<0.01).
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Figure 4: Deletion IFT20 in double-negative thymocytes affected the development of CIA. (a) Timeline for the CIA mouse model. Seven- to eight-week-old mice were immunized with 100 μL emulsion containing equal volume of CFA (including 5 mg·mL−1 of M. tuberculosis) and chicken type II collagen solution (2 mg·mL−1 in 0.05 mol·mL−1 acetic acid). The booster injection (Insert site: 3 cm from the base of the tail Injection site: 1.5 cm from the base) was made with the same emulsions 21 days after initial injection (Insert site: 2 cm from the base of the tail Injection site: 0.5 cm from the base). Arthritis usually can be observed 4–6 weeks after the first immunization with the maximum incidence of arthritis at 6–7 weeks. All the experimental mice were harvested at day 66 for the histological analysis. (b) Mean clinical scores±s.d. of arthritis in Lck-Cre/IFT20+/+ (n=6) and Lck-Cre/IFT20f/f mice (n=4) with arthritis (P<0.01 after day 40). (c) Percentage of mice of Lck-Cre/IFT20+/+ and Lck-Cre/IFT20f/f mice that developed arthritis (n=10, P<0.05 after day 40). (d) The maximum clinical score was recorded until day 66 (n=10, *P<0.01). (e) Hind paws of type II collagen immunized Lck-Cre/IFT20+/+ and Lck-Cre/ IFT20f/f mice. (f) The hind paw thickness was measured at day 66 to indicate arthritis development (n=10, *P<0.01).

Mentions: To determine the functional role of IFT20 in T cell-mediated inflammatory diseases, we created the CIA model in wild-type and IFT20 mutant mice. The C57/BL6 strain has been considered to be a CIA-resistant strain, due to little or no incidence of CIA upon immunization.18,40 However, Campbell et al.27 successfully induced arthritis at relatively high incidence in CIA-resistant mouse strains, such as C57BL/ 6, C57BL/10, and 129/Sv mice (H-2b) with CFA, which contains 5 mg·mL−1 of M. tuberculosis. We used this CIA model with a slight modification, and the immunization schedule followed is shown in Figure 4a. Six- to seven-week-old, age-matched Lck-Cre/IFT20+/+ and Lck-Cre/IFT20f/f C57BL/6 mice were chosen for generating the CIA model. Stable emulsions with an equal volume of CFA (containing 5 mg·mL−1 of M. tuberculosis) and chicken type II collagen solution (2 mg·mL−1 in 0.05 mol·mL−1 acetic acid) were prepared immediately before the injection on day 0 (initial immunization) and day 21 (booster injection). Each mouse received an accurate 0.1 mL of the emulsion each time. For the initial immunization, the injection site was 2 cm from the base of the tail, and a 25 gauge×5/8″ needle was used to reach to 0.5 cm from the base for injection. The booster injection was inserted at 3 cm from the base of the tail until the needle tip reached 1.5 cm from the base (Figure 4a). The needle was wiped and inserted bevel up and parallel to the tail in order to prevent leakage of emulsion. We observed arthritis at week 5 after the first immunization. The maximum incidence of arthritis was achieved at 6–7 weeks. To quantitatively evaluate the severity of the arthritis, four paws were observed and measured every 3 days up to 66 days after the first immunization using the grades as described in the section on ‘Materials and methods’. The incidence of CIA was significantly lower in the Lck-Cre/ IFT20f/f group than that in the Lck-Cre/IFT20+/+ group (Figure 4c). Even during development of the arthritis, the severity of the disease in Lck-Cre/IFT20f/f mice was much less than that in Lck-Cre/IFT20+/+ mice (Figure 4b and 4c). The hind paw of Lck-Cre/IFT20+/+ displayed severe joint inflammation evidenced by marked swelling and erythema of paws (Figure 4e). In contrast, Lck-Cre/IFT20f/f mice were resistant to developing CIA and showed no signs or only slight signs of paw and/or joint swelling (Figure 4e). The thickness of hind paws was measured at the end of the experiment. Lck-Cre/IFT20f/f mice had significantly thinner paws than Lck-Cre/IFT20+/+ mice, which confirmed that the development and severity of CIA was reduced in Lck-Cre/ IFT20f/f mice (Figure 4f).


Deletion of IFT20 in early stage T lymphocyte differentiation inhibits the development of collagen-induced arthritis.

Yuan X, Garrett-Sinha LA, Sarkar D, Yang S - Bone Res (2014)

Deletion IFT20 in double-negative thymocytes affected the development of CIA. (a) Timeline for the CIA mouse model. Seven- to eight-week-old mice were immunized with 100 μL emulsion containing equal volume of CFA (including 5 mg·mL−1 of M. tuberculosis) and chicken type II collagen solution (2 mg·mL−1 in 0.05 mol·mL−1 acetic acid). The booster injection (Insert site: 3 cm from the base of the tail Injection site: 1.5 cm from the base) was made with the same emulsions 21 days after initial injection (Insert site: 2 cm from the base of the tail Injection site: 0.5 cm from the base). Arthritis usually can be observed 4–6 weeks after the first immunization with the maximum incidence of arthritis at 6–7 weeks. All the experimental mice were harvested at day 66 for the histological analysis. (b) Mean clinical scores±s.d. of arthritis in Lck-Cre/IFT20+/+ (n=6) and Lck-Cre/IFT20f/f mice (n=4) with arthritis (P<0.01 after day 40). (c) Percentage of mice of Lck-Cre/IFT20+/+ and Lck-Cre/IFT20f/f mice that developed arthritis (n=10, P<0.05 after day 40). (d) The maximum clinical score was recorded until day 66 (n=10, *P<0.01). (e) Hind paws of type II collagen immunized Lck-Cre/IFT20+/+ and Lck-Cre/ IFT20f/f mice. (f) The hind paw thickness was measured at day 66 to indicate arthritis development (n=10, *P<0.01).
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Figure 4: Deletion IFT20 in double-negative thymocytes affected the development of CIA. (a) Timeline for the CIA mouse model. Seven- to eight-week-old mice were immunized with 100 μL emulsion containing equal volume of CFA (including 5 mg·mL−1 of M. tuberculosis) and chicken type II collagen solution (2 mg·mL−1 in 0.05 mol·mL−1 acetic acid). The booster injection (Insert site: 3 cm from the base of the tail Injection site: 1.5 cm from the base) was made with the same emulsions 21 days after initial injection (Insert site: 2 cm from the base of the tail Injection site: 0.5 cm from the base). Arthritis usually can be observed 4–6 weeks after the first immunization with the maximum incidence of arthritis at 6–7 weeks. All the experimental mice were harvested at day 66 for the histological analysis. (b) Mean clinical scores±s.d. of arthritis in Lck-Cre/IFT20+/+ (n=6) and Lck-Cre/IFT20f/f mice (n=4) with arthritis (P<0.01 after day 40). (c) Percentage of mice of Lck-Cre/IFT20+/+ and Lck-Cre/IFT20f/f mice that developed arthritis (n=10, P<0.05 after day 40). (d) The maximum clinical score was recorded until day 66 (n=10, *P<0.01). (e) Hind paws of type II collagen immunized Lck-Cre/IFT20+/+ and Lck-Cre/ IFT20f/f mice. (f) The hind paw thickness was measured at day 66 to indicate arthritis development (n=10, *P<0.01).
Mentions: To determine the functional role of IFT20 in T cell-mediated inflammatory diseases, we created the CIA model in wild-type and IFT20 mutant mice. The C57/BL6 strain has been considered to be a CIA-resistant strain, due to little or no incidence of CIA upon immunization.18,40 However, Campbell et al.27 successfully induced arthritis at relatively high incidence in CIA-resistant mouse strains, such as C57BL/ 6, C57BL/10, and 129/Sv mice (H-2b) with CFA, which contains 5 mg·mL−1 of M. tuberculosis. We used this CIA model with a slight modification, and the immunization schedule followed is shown in Figure 4a. Six- to seven-week-old, age-matched Lck-Cre/IFT20+/+ and Lck-Cre/IFT20f/f C57BL/6 mice were chosen for generating the CIA model. Stable emulsions with an equal volume of CFA (containing 5 mg·mL−1 of M. tuberculosis) and chicken type II collagen solution (2 mg·mL−1 in 0.05 mol·mL−1 acetic acid) were prepared immediately before the injection on day 0 (initial immunization) and day 21 (booster injection). Each mouse received an accurate 0.1 mL of the emulsion each time. For the initial immunization, the injection site was 2 cm from the base of the tail, and a 25 gauge×5/8″ needle was used to reach to 0.5 cm from the base for injection. The booster injection was inserted at 3 cm from the base of the tail until the needle tip reached 1.5 cm from the base (Figure 4a). The needle was wiped and inserted bevel up and parallel to the tail in order to prevent leakage of emulsion. We observed arthritis at week 5 after the first immunization. The maximum incidence of arthritis was achieved at 6–7 weeks. To quantitatively evaluate the severity of the arthritis, four paws were observed and measured every 3 days up to 66 days after the first immunization using the grades as described in the section on ‘Materials and methods’. The incidence of CIA was significantly lower in the Lck-Cre/ IFT20f/f group than that in the Lck-Cre/IFT20+/+ group (Figure 4c). Even during development of the arthritis, the severity of the disease in Lck-Cre/IFT20f/f mice was much less than that in Lck-Cre/IFT20+/+ mice (Figure 4b and 4c). The hind paw of Lck-Cre/IFT20+/+ displayed severe joint inflammation evidenced by marked swelling and erythema of paws (Figure 4e). In contrast, Lck-Cre/IFT20f/f mice were resistant to developing CIA and showed no signs or only slight signs of paw and/or joint swelling (Figure 4e). The thickness of hind paws was measured at the end of the experiment. Lck-Cre/IFT20f/f mice had significantly thinner paws than Lck-Cre/IFT20+/+ mice, which confirmed that the development and severity of CIA was reduced in Lck-Cre/ IFT20f/f mice (Figure 4f).

Bottom Line: Recently, IFT20 was found to be also expressed in non-ciliated T cells and have functions in immune synapse formation and signaling in vitro.However, how IFT20 regulates T-cell development and activation in vivo is still unknown.These results demonstrate that deletion of IFT20 in the early stage of T-cell development inhibited CIA development through regulating T-cell development and the expression of critical cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, School of Dental Medicine, University at Buffalo, The State University of New York , Buffalo, NY 14214, USA.

ABSTRACT
IFT20 is the smallest member of the intraflagellar transport protein (IFT) complex B. It is involved in cilia formation. Studies of IFT20 have been confined to ciliated cells. Recently, IFT20 was found to be also expressed in non-ciliated T cells and have functions in immune synapse formation and signaling in vitro. However, how IFT20 regulates T-cell development and activation in vivo is still unknown. We deleted the IFT20 gene in early and later stages of T-cell development by crossing IFT20(flox/flox) (IFT20(f/f) ) mice with Lck-Cre and CD4-Cre transgenic mice, and investigated the role of IFT20 in T-cell maturation and in the development of T cell-mediated collagen-induced arthritis (CIA). We found that both Lck-Cre/IFT20(f/f) and CD4-Cre/IFT20(f/f) mice were indistinguishable from their wild-type littermates in body size, as well as in the morphology and weight of the spleen and thymus. However, the number of CD4- and CD8-positive cells was significantly lower in thymus and spleen in Lck-Cre/IFT20(f/f) mice. Meanwhile, the incidence and severity of CIA symptoms were significantly decreased, and inflammation in the paw was significantly inhibited in Lck-Cre/IFT20(f/f) mice compared to Lck-Cre/IFT20(+/+) littermates. Deletion IFT20 in more mature T cells of CD4-Cre/IFT20(f/f) mice had only mild effects on the development of T cells and CIA. The expression of IL-1β, IL-6 and TGF-β1 were significantly downregulated in the paw of Lck-Cre/IFT20(f/f) mice, but just slight decreased in CD4-Cre/IFT20(f/f) mice. These results demonstrate that deletion of IFT20 in the early stage of T-cell development inhibited CIA development through regulating T-cell development and the expression of critical cytokines.

No MeSH data available.


Related in: MedlinePlus