Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms

Abstract

Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4+CD25High T lymphocytes (Tregs) and cytotoxic CD4+CD25- T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4+CD25HighIL-4+, CD4+CD25HighIL-10+ and CD4+CD25HighTGF-β+ Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Mutant SOD1 Tregs suppressed activation of adult mSOD1 microglia (Mc). (a–b) Mc were isolated from 130 day old mSOD1 mice or wild-type (WT) mice. After 2 days in culture, mSOD1 Mc expressed more NOX2 mRNA than WT Mc (a). NOX2 protein was also up-regulated in mSOD1 Mc compared with WT Mc; β-actin was used to control protein loading. Although more WT Mc protein was intentionally loaded onto the gel as indicated by the β-actin signal, NOX2 protein from WT Mc was minimally detectable. Semi-quantification of NOX2 protein was performed by analyzing band intensities after normalization to β-actin (b). (c) iNOS from WT Mc was not detectable; iNOS was upregulated in mSOD1 Mc when assayed by quantitative RT-PCR. β-actin was used as a internal control. (d) Nitrite+nitrate levels were used as an index of nitric oxide (NO) production and were measured in the supernatants of WT or mSOD1 Mc co-cultures at 2 days post-plating. mSOD1 Mc produced more NO than WT Mc. (e–g) Tregs and Teffs were purified from 100 day old mSOD1 mice, and then co-cultured with mSOD1 Mc for 2 days. mSOD1 Tregs inhibited NOX2 (e), iNOS (f) expression, and NO production (g) in mSOD1 Mc compared with mSOD1 Teffs and Mc co-cultures; Tregs and Teffs expressed minimal NOX2, iNOS and NO. Data shown as mean±SE of 4–5 independent experiments with duplicate or triplicate wells. *p&&p<0.01 vs. WT Mc+WT Teffs; #p<0.05, ##p<0.01 vs. mSOD1 Mc+ mSOD1 Teffs. Mc = microglia; Tc = T cells; WT = wild-type.

Fig. 1

Mutant SOD1 Tregs suppressed activation of adult mSOD1 microglia (Mc). (a–b) Mc were isolated from 130 day old mSOD1 mice or wild-type (WT) mice. After 2 days in culture, mSOD1 Mc expressed more NOX2 mRNA than WT Mc (a). NOX2 protein was also up-regulated in mSOD1 Mc compared with WT Mc; β-actin was used to control protein loading. Although more WT Mc protein was intentionally loaded onto the gel as indicated by the β-actin signal, NOX2 protein from WT Mc was minimally detectable. Semi-quantification of NOX2 protein was performed by analyzing band intensities after normalization to β-actin (b). (c) iNOS from WT Mc was not detectable; iNOS was upregulated in mSOD1 Mc when assayed by quantitative RT-PCR. β-actin was used as a internal control. (d) Nitrite+nitrate levels were used as an index of nitric oxide (NO) production and were measured in the supernatants of WT or mSOD1 Mc co-cultures at 2 days post-plating. mSOD1 Mc produced more NO than WT Mc. (e–g) Tregs and Teffs were purified from 100 day old mSOD1 mice, and then co-cultured with mSOD1 Mc for 2 days. mSOD1 Tregs inhibited NOX2 (e), iNOS (f) expression, and NO production (g) in mSOD1 Mc compared with mSOD1 Teffs and Mc co-cultures; Tregs and Teffs expressed minimal NOX2, iNOS and NO. Data shown as mean±SE of 4–5 independent experiments with duplicate or triplicate wells. *p&&p<0.01 vs. WT Mc+WT Teffs; #p<0.05, ##p<0.01 vs. mSOD1 Mc+ mSOD1 Teffs. Mc = microglia; Tc = T cells; WT = wild-type.

Fig. 2

mSOD1 Tregs released IL-4 protein. Tregs or Teffs isolated from mSOD1 or wild-type (WT) mice were co-cultured with mSOD1 or WT microglia (Mc) for 2 days. IL-4 protein was measured in the supernatant of each co-culture. Increased IL-4 protein levels were observed in mSOD1 Tregs and microglia co-cultures. IL-4 was not detectable in microglia alone cultures. Data shown as mean±SE of 4–5 independent experiments with duplicate or triplicate wells. **p##p<0.01 vs. WT Mc+ WT Tregs. Mc = microglia; Tc = T cells; WT = wild-type.

Fig. 3

IL-4 released by mSOD1 Tregs suppressed microglial (Mc) activation. (a–b) A IL-4 blocking antibody (150 ng/ml) was added to mSOD1 Mc+Teffs or mSOD1 Mc+Tregs co-cultures. After 2 days of incubation, RNAs were extracted for quantitative RT-PCR analyses and supernatants were collected for detecting nitrite and nitrate levels as an indicator of nitric oxide (NO) production. IL-4 blocking antibody reversed NOX2 mRNA (a), iNOS mRNA (b), and NO production (c) in mSOD1 Mc+Tregs co-cultures. (d) Conditioned-media (CM) of mSOD1 Teffs or Tregs were used to culture mSOD1 Mc for 2 days. CM of mSOD1 Tregs suppressed microglial NOX2 mRNA, and IL-4 neutralizing antibody blocked the suppression of Tregs CM. (e) Different doses of IL-4 protein (0.01–10ng/ml) were added to mSOD1 Mc cultures. IL-4 protein inhibited NOX2 of mSOD1 Mc in a dose-dependent manner. Data shown as mean±SE of 3–5 independent experiments with duplicate or triplicate wells. #p<0.05, ##p<0.01 vs. mSOD1 Mc+ mSOD1 Teffs; &&p<0.01 vs. Mc+Tregs; **p<0.01 vs. mSOD1 Mc; ‡‡p<0.01 vs. mSOD1 Mc in CM of mSOD1 Teffs; §p<0.05 vs. mSOD1 Mc in CM of mSOD1 Tregs without IL-4 Ab. Ab = antibody; CM: Conditioned-media; Mc = microglia; Tc = T cells.

Fig. 3

IL-4 released by mSOD1 Tregs suppressed microglial (Mc) activation. (a–b) A IL-4 blocking antibody (150 ng/ml) was added to mSOD1 Mc+Teffs or mSOD1 Mc+Tregs co-cultures. After 2 days of incubation, RNAs were extracted for quantitative RT-PCR analyses and supernatants were collected for detecting nitrite and nitrate levels as an indicator of nitric oxide (NO) production. IL-4 blocking antibody reversed NOX2 mRNA (a), iNOS mRNA (b), and NO production (c) in mSOD1 Mc+Tregs co-cultures. (d) Conditioned-media (CM) of mSOD1 Teffs or Tregs were used to culture mSOD1 Mc for 2 days. CM of mSOD1 Tregs suppressed microglial NOX2 mRNA, and IL-4 neutralizing antibody blocked the suppression of Tregs CM. (e) Different doses of IL-4 protein (0.01–10ng/ml) were added to mSOD1 Mc cultures. IL-4 protein inhibited NOX2 of mSOD1 Mc in a dose-dependent manner. Data shown as mean±SE of 3–5 independent experiments with duplicate or triplicate wells. #p<0.05, ##p<0.01 vs. mSOD1 Mc+ mSOD1 Teffs; &&p<0.01 vs. Mc+Tregs; **p<0.01 vs. mSOD1 Mc; ‡‡p<0.01 vs. mSOD1 Mc in CM of mSOD1 Teffs; §p<0.05 vs. mSOD1 Mc in CM of mSOD1 Tregs without IL-4 Ab. Ab = antibody; CM: Conditioned-media; Mc = microglia; Tc = T cells.

Fig. 4

FACS followed by flow cytometric analyses revealed mSOD1 CD4+CD25HighFoxp3+ Tregs produce IL-4. (a) CD4+CD25High Tregs were isolated from 100 day old mSOD1 mice using mouse regulatory T cell isolation kits from Miltenyi Biotec. Flow cytometry data showed that 80% of CD4+CD25High Tregs express Foxp3. (b–c) Highly purified CD4+CD25HighFoxp3(GFP)+ Tregs were sorted from 100 day old Foxp3(GFP)+/mSOD1G93A mice by FACS. After 1 day incubation, CD4+CD25HighFoxp3(GFP)+ Tregs were assayed for IL-4 detection by flow cytometry. CD4+CD25HighFoxp3+IL-4+ Tregs accounted for 6.1% in Tregs alone cultures (b). When co-cultured with microglia (Mc) for 1 day, more CD4+CD25HighFoxp3+IL-4+ Tregs (12.0%) were observed (c) than mSOD1 Tregs alone cultures (b). Mc = microglia.

Fig. 5

The suppressive effects of mSOD1 Tregs on microglial (Mc) activation was not dependent on IL-10, TGF-β, or CTLA-4. mSOD1 Mc were co-cultured with mSOD1 Teffs or mSOD1 Tregs for 2 days with or without blocking antibodies to IL-4, IL-10, TGF-β, or CTLA-4. IL-4 neutralizing antibody reversed microglial NOX2 mRNA expression in mSOD1 Mc and Tregs co-cultures. However, the addition of IL-10, TGF-β, or CTLA-4 neutralizing antibodies did not change mRNA levels of NOX2 in mSOD1 Mc and Tregs co-cultures. Data shown as mean±SE of three independent experiments with duplicate or triplicate wells. ##p<0.01 vs. Mc+Teffs without blocking antibody; &&p<0.01 vs. Mc+Tregs without blocking antibody. Ab = antibody; Mc = microglia.

Fig. 6

Tregs isolated from mSOD1 mice during the rapidly progressing phase suppressed mSOD1 microglial (Mc) activation. Tregs from slowly progressing phase (100 day old) or from rapidly progressing phase (160 day old) mSOD1 mice were co-cultured with mSOD1 Mc for 2 days. Both 100 day Tregs and 160 day Tregs equally suppressed microglial NOX2 mRNA levels. Data shown as mean±SE of at least three independent experiments with duplicate or triplicate wells. **p##p<0.01 vs. Mc +Teffs in each group. WT = wild-type; Mc = microglia.

Fig. 7

Numbers of CD4+CD25HighFoxp3+ Tregs, IL-4-, TGF-β-, and IL-10-expressing CD4+CD25HighFoxp3+ Tregs were different in wild-type (WT) mice (100 day old), slowly progressing mSOD1 mice (100 day old), and rapidly progressing mSOD1 mice (160 day old). The Tregs were isolated from the lymph nodes (axillary, inguinal and superficial cervical) of WT or mSOD1 mice, and analyzed by flow cytometry. (a) More CD4+CD25HighFoxp3+ Tregs were present in 100 day mSOD1 mice than WT mice, while less CD4+CD25HighFoxp3+ Tregs were found in 160 day mSOD1 mice compared with 100 day mSOD1 mice. (b–d) Expression of IL-4, TGF-β, and IL-10 were also analyzed in CD4+CD25HighFoxp3+ Tregs populations. The numbers of CD4+CD25HighFoxp3+IL-4+ Tregs (b), CD4+CD25HighFoxp3+TGF-β+ Tregs (c), CD4+CD25HighFoxp3+IL-10+ Tregs (d) were increased in 100 day old mSOD1 mice, whereas these cytokines were decreased in 160 day old mSOD1 mice. Data shown as mean±SE of three independent experiments. *p<0.05, **p<0.01 vs. WT mice; #p<0.05, ##p<0.01 vs. 100 day mSOD1 mice. WT = wild-type.

Fig. 8

Proliferation and cytotoxicity of Teffs were different among wild-type (WT) mice (100 day old), slowly progressing mSOD1 mice (100 day old), and rapidly progressing mSOD1 mice (160 day old). (a) Purified Tregs and Teffs were cultured for 3 days and cell proliferation was measured. Teffs from 100 day old mSOD1 mice proliferated slower than WT Teffs, and Teffs from 160 day old mSOD1 mice (160 day old) proliferated faster than Teffs from 100 day old mSOD1 mice. (b) IFN-γ mRNA was measured as an index of Teffs cytotoxicity. One hundred day mSOD1 Teffs expressed less IFN-γ than WT Teffs; 160 day mSOD1 Teffs expressed more IFN-γ than 100 day mSOD1 Teffs. Tregs did not proliferate. Data shown as mean±SE of three independent experiments with duplicate or triplicate wells. *p&&p<0.01 vs. 100 day mSOD1 Teffs. WT = wild-type.

Fig. 9

Inhibition of Teffs by 100 day mSOD1 Tregs is dependent on IL-4, IL-10, and TGF-β. Teffs were co-cultured with corresponding Tregs for 3 days with or without blocking antibodies to IL-4, IL-10, and TGF-β. Teffs proliferation was measured and suppressive activity of Tregs on Teffs was calculated. (a) The inhibition rate of 100 day or 160 day mSOD1 Tregs on mSOD1 Teffs was similar as the rate of wild-type (WT) Tregs on WT Teffs. The combination of neutralizing antibodies to IL-4, IL-10, and TGF-β decreased the inhibition rate of 100 day mSOD1 Tregs, but these 3 antibodies together did not change the inhibition rate of WT Tregs on WT Teffs or 160 day old Tregs on 160 day Teffs. Neither two of these 3 antibodies blocked the suppressive effect of 100 day, 160 day mSOD1 Tregs or WT Tregs. (b) One hundred day mSOD1 Tregs or 160 day mSOD1 Tregs inhibited IFN-γ mRNA in Teffs to the similar levels as those in WT Teffs and Tregs co-cultures. The addition of neutralizing antibodies to IL-4, IL-10, and TGF-β increased IFN-γ expression in 100 day mSOD1 Teffs and Tregs co-cultures, but did not change the levels of IFN-γ in either WT Teffs + Tregs or 160 day mSOD1 Teffs + Tregs co-cultures. Data shown as mean±SE of three independent experiments with duplicate or triplicate wells. *p&p<0.05, vs. 100 day mSOD1 Teffs; ##p<0.01 vs. Teffs in each group; ‡p<0.05, ‡‡p<0.01 vs. 100 day mSOD1 Teffs + Tregs without blocking antibodies; Δp<0.05, ΔΔp<0.01 vs. WT Teffs + Tregs + 3 blocking antibodies. Ab = antibody; Tc = T cells; WT = wild-type.