A ) Representative pictures of WT hippocampal neurons transfected with the SBP - mCherry - GluK2 RUSH construct , followed by treating neurons with biotin and anti - SBP antibodies ( mouse , green ) for 20 min or 40 min to live - label the GluK2 transported to the neuronal cell surface . As a control ( no biotin ) , neurons were incubated only with SBP antibody for 40 min . The surface / total ratio of SBP - mCherry - GluK2 constructs was estimated by calculating the ratio of SBP ( green ) / mCherry ( red ) fluorescence signals in the same area chosen for quantification . B ) Quantification results for A , indicating that incubation with biotin increases the surface / total ratio of SBP - mCherry - GluK2 over time . No Biotin n = 16 , 20 min n = 13 , 40 min n = 15 , all biological replicates . Graph shows mean ± SEM , 1 - way ANOVA p < 0 . 0001 and Dunnett ' s post hoc test , No Biotin vs 20 min * p = 0 . 0030 ; No Biotin vs 40 min * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

A) Representative pictures of WT hippocampal neurons transfected with the SBP-mCherry-GluK2 RUSH construct, followed by treating neurons with biotin and anti-SBP antibodies (mouse, green) for 20 min or 40 min to live-label the GluK2 transported to the neuronal cell surface. As a control (no biotin), neurons were incubated only with SBP antibody for 40 min. The surface/total ratio of SBP-mCherry-GluK2 constructs was estimated by calculating the ratio of SBP (green)/ mCherry (red) fluorescence signals in the same area chosen for quantification. B) Quantification results for A, indicating that incubation with biotin increases the surface/total ratio of SBP-mCherry-GluK2 over time. No Biotin n=16, 20 min n=13, 40 min n=15, all biological replicates. Graph shows mean±SEM, 1-way ANOVA p<0.0001 and Dunnett's post hoc test, No Biotin vs 20 min *p=0.0030; No Biotin vs 40 min ***p<0.0001.


C - D ) SEZ6 flox / flox neurons were lentivirally infected with inactivated Cre ( Y324F , Ctrl ) or Cre recombinase ( SEZ6KO ) , followed by transfection with the SBP - mCherry - GluK2 construct and treated with biotin and anti - SBP antibodies ( mouse , green ) for 20 min . Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti - Cre antibody ( rabbit , blue ) . SEZ6KO neurons showed a significantly reduced surface / total ratio of SBP - mCherry - GluK2 ( ctl n = 25 , SEZ6KO n = 23 biological replicates . Graph shows mean ± SEM , unpaired t - test , * * p = 0 . 0068 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D) SEZ6 flox/flox neurons were lentivirally infected with inactivated Cre (Y324F, Ctrl) or Cre recombinase (SEZ6KO), followed by transfection with the SBP-mCherry-GluK2 construct and treated with biotin and anti-SBP antibodies (mouse, green) for 20 min. Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti-Cre antibody (rabbit, blue). SEZ6KO neurons showed a significantly reduced surface/total ratio of SBP-mCherry-GluK2 (ctl n=25, SEZ6KO n=23 biological replicates. Graph shows mean ± SEM, unpaired t-test, **p=0.0068).


B , SEZ6 constitutive KO neurons were transduced with SEZ6 constructs . SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature ( higher ) and immature ( lower ) forms of SEZ6 . No difference in the apparent molecular weight was detected because the high percentage of the gel used ( 12 % ) and the small C - terminal deletion ( 39 amino acids ) . SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF ( with HA and Flag - tags ) was detected at the expected low molecular weight . Additionally , the C - terminal fragment generated from SEZ6FL by BACE1 cleavage was detected . Due to its lack of an HA - tag , it had a lower molecular weight than SEZ6CTF . The C - terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight . GluK2 / 3 glycosylation was rescued when SEZ6FL , SEZ6Δcyto and SEZ6ecto were expressed in the neurons , but SEZ6CTF was not able to rescue the phenotype . Color coding for asterisks is the same as in figure 4A . Representative blot of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, SEZ6 constitutive KO neurons were transduced with SEZ6 constructs. SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature (higher) and immature (lower) forms of SEZ6. No difference in the apparent molecular weight was detected because the high percentage of the gel used (12%) and the small C-terminal deletion (39 amino acids). SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF (with HA and Flag-tags) was detected at the expected low molecular weight. Additionally, the C-terminal fragment generated from SEZ6FL by BACE1 cleavage was detected. Due to its lack of an HA-tag, it had a lower molecular weight than SEZ6CTF. The C-terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight. GluK2/3 glycosylation was rescued when SEZ6FL, SEZ6Δcyto and SEZ6ecto were expressed in the neurons, but SEZ6CTF was not able to rescue the phenotype. Color coding for asterisks is the same as in figure 4A. Representative blot of 3 independent experiments.


C ) SEZ6ecto was present as the mature , secreted band in the conditioned medium in panel C .

C) SEZ6ecto was present as the mature, secreted band in the conditioned medium in panel C.


D ) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto . SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6 , as expected for a protein retained in the ER . SEZ6Δcyto , but not SEZ6ΔcytoER , was able to rescue GluK2 / 3 glycosylation , showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2 / 3 glycosylation ( n = 5 ) . Color coding for asterisks is the same as in figure 4A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto. SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6, as expected for a protein retained in the ER. SEZ6Δcyto, but not SEZ6ΔcytoER, was able to rescue GluK2/3 glycosylation, showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2/3 glycosylation (n=5). Color coding for asterisks is the same as in figure 4A.


E ) HEK293T cells were co - transfected with a GluK2a plasmid and the SEZ6 constructs . Expression of SEZ6 constructs was analyzed in the total lysates ( " Input " ) and revealed a similar expression of the different proteins . GluK2a was immunoprecipitated with GluK2 / 3 antibody and the SEZ6 mutants were detected with the HA . 11 antibody ( " IP " ) . SEZ6FL , SEZ6Δcyto and SEZ6ecto , but not SEZ6CTF were co - immunoprecipitated with GluK2a , suggesting that SEZ6 extracellular domain is necessary for GluK2a binding . Representative blot of 2 independent experiments . * indicates the light chain of the antibody used for immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) HEK293T cells were co-transfected with a GluK2a plasmid and the SEZ6 constructs. Expression of SEZ6 constructs was analyzed in the total lysates ("Input") and revealed a similar expression of the different proteins. GluK2a was immunoprecipitated with GluK2/3 antibody and the SEZ6 mutants were detected with the HA.11 antibody ("IP"). SEZ6FL, SEZ6Δcyto and SEZ6ecto, but not SEZ6CTF were co-immunoprecipitated with GluK2a, suggesting that SEZ6 extracellular domain is necessary for GluK2a binding. Representative blot of 2 independent experiments. * indicates the light chain of the antibody used for immunoprecipitation.


A Ex vivo flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + IL - 2 , KLH + RA , KLH + RA + IL - 2 . Results are representative FACS plots of LAG3 + CD4 + or CD25 + Foxp3 + cells after gating on live single CD3 + CD4 + CD49b + Foxp3 - and live single CD3 + CD4 + lymphocytes respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+ lymphocytes respectively.


A Ex vivo flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + IL - 2 , KLH + RA , KLH + RA + IL - 2 . Results are representative FACS plots of LAG3 + CD4 + or CD25 + Foxp3 + cells after gating on live single CD3 + CD4 + CD49b + Foxp3 - and live single CD3 + CD4 +

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+


C Expression of Tr1 markers PD - 1 , CD226 , ICOS , CD25 and CTLA - 4 on CD49b + LAG - 3 + Foxp3 - CD4 + T cells or on CD49b - CD4 + T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Expression of Tr1 markers PD-1, CD226, ICOS, CD25 and CTLA-4 on CD49b+ LAG-3+ Foxp3-CD4+ T cells or on CD49b-CD4+ T cells.


D IL - 10 , IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + RA , KLH + IL - 2 , KLH + RA + IL - 2 or KLH + LP - GMP ( LP1569 + c - di - GMP ) and cultured for 5 days with increasing concentrations of KLH . Bars are mean + SEM ( n = 4 or 5 ) . * * p < 0 . 01 , * * * p < 0 . 001 , KLH + IL - 2 + RA vs KLH , # # p < 0 . 01 , # # # p < 0 . 001 KLH + IL - 2 + RA vs KLH + RA , † † † p < 0 . 001 KLH + IL - 2 + RA vs KLH + IL - 2 by two - way ANOVA and Tukey post hoc test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

D IL-10, IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + RA, KLH + IL-2, KLH + RA + IL-2 or KLH + LP-GMP (LP1569 + c-di-GMP) and cultured for 5 days with increasing concentrations of KLH. Bars are mean + SEM (n=4 or 5). **p<0.01, ***p<0.001, KLH + IL-2 + RA vs KLH, ##p<0.01, ###p <0.001 KLH + IL-2 + RA vs KLH + RA, †††p<0.001 KLH + IL-2 + RA vs KLH + IL-2 by two-way ANOVA and Tukey post hoc test.


A Flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , MOG + DMSO ( MOG ) , MOG + RA + IL - 2 . Cells were gated on live single CD3 + lymphocytes . FMO : Fluorescence Minus One . B Mean percentages of IL - 10 + , LAG - 3 + , PD1 + or Foxp3 + CD25 + CD4 T cells as described in A . Cells were gated on live single CD3 + lymphocytes ( top left panel ) or on live single CD3 + CD4 + lymphocytes . Bars are mean + SEM ( n = 5 ) . * p < 0 . 05 , * * * p < 0 . 01 by one - way ANOVA and Tukey post hoc test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), MOG + DMSO (MOG), MOG + RA + IL-2. Cells were gated on live single CD3+ lymphocytes. FMO: Fluorescence Minus One. B Mean percentages of IL-10+, LAG-3+, PD1+ or Foxp3+ CD25+ CD4 T cells as described in A. Cells were gated on live single CD3+ lymphocytes (top left panel) or on live single CD3+ CD4+ lymphocytes. Bars are mean + SEM (n=5). *p <0.05, ***p <0.01 by one-way ANOVA and Tukey post hoc test.


C EAE clinical scores in mice immunized s . c . with MOG + RA , MOG + IL - 2 , MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 12 , except for MOG + IL - 2 , where n = 6 ; combined from two experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , MOG + IL - 2 + RA vs Veh , # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 , MOG + IL - 2 + RA vs MOG + RA , † p < 0 . 05 , † † p < 0 . 01 , † † † p < 0 . 001 , MOG + IL - 2 + RA vs MOG + IL - 2 by two - way ANOVA and Tukey post hoc test .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

C EAE clinical scores in mice immunized s.c. with MOG + RA, MOG + IL-2, MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=12, except for MOG + IL-2, where n=6; combined from two experiments). *p<0.05, **p<0.01, ***p <0.001, MOG + IL-2 + RA vs Veh, #p<0.05, ##p<0.01, ###p<0.001, MOG + IL-2 + RA vs MOG + RA, †p<0.05, ††p<0.01, †††p <0.001, MOG + IL-2 + RA vs MOG + IL-2 by two-way ANOVA and Tukey post hoc test.


D Representative FACS plots of MOG dextramer + CD4 T cells in LNs of naïve mice or 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Representative FACS plots of MOG dextramer+ CD4 T cells in LNs of naïve mice or 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+.


E Mean percentages of MOG dextramer + CD4 T cells in LNs 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Mean percentages of MOG dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+. Bars are mean + SEM (n=5 or 6). *p <0.05 by unpaired two-tailed t test.


F Mean fluorescence intensity ( MFI ) of CD49b , ICOS and PD - 1 by MOG - dextramer + CD4 T cells in LNs 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + dextramer + . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 , * * p < 0 . 01 by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Mean fluorescence intensity (MFI) of CD49b, ICOS and PD-1 by MOG-dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+ dextramer+. Bars are mean + SEM (n=5 or 6). *p <0.05, **p <0.01 by unpaired two-tailed t test.


G MOG - specific IL - 17 and IFN - γ production by spleen cells from day 7 of EAE , quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG . Bars are mean + SEM ( n = 3 or 4 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - ANOVA and Tukey post hoc test .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G MOG-specific IL-17 and IFN-γ production by spleen cells from day 7 of EAE, quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG. Bars are mean + SEM (n=3 or 4). *p<0.05, **p<0.01 and ***p<0.001 by two-ANOVA and Tukey post hoc test.


A Representative FACS plots of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 or vehicle ( Veh ) only . Cells were gated as single live CD45 + CD3 + CD4 + . B Mean absolute numbers of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 . Bars are mean + SEM ( n = 4 or 5 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+. B Mean absolute numbers of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2. Bars are mean + SEM (n=4 or 5). *p<0.05 by unpaired two-tailed t test.


A Representative FACS plots of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 or vehicle ( Veh ) only . Cells were gated as single live CD45 + CD3 + CD4 + .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+.


C Representative FACS plots of CD45 + CD4 T cells in the spinal cords of mice on day 12 of EAE . Cells were gated as single live CD45 + . D Mean absolute numbers of infiltrating CD45 + cells in the spinal cords of mice on day 12 of EAE . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative FACS plots of CD45+ CD4 T cells in the spinal cords of mice on day 12 of EAE. Cells were gated as single live CD45+. D Mean absolute numbers of infiltrating CD45+ cells in the spinal cords of mice on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.


C Representative FACS plots of CD45 + CD4 T cells in the spinal cords of mice on day 12 of EAE . Cells were gated as single live CD45 + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative FACS plots of CD45+ CD4 T cells in the spinal cords of mice on day 12 of EAE. Cells were gated as single live CD45+.


E Representative FACS plots of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + . F Mean absolute numbers of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Representative FACS plots of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+. F Mean absolute numbers of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.


E Representative FACS plots of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Representative FACS plots of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.


G Representative histogram of LAG - 3 expression on CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Representative histogram of LAG-3 expression on CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.


H Inverse correlation between LAG - 3 and IL - 17 and IFN - γ expression on CD4 T cells in the spinal cords on day 12 of EAE . * p < 0 . 05 by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Inverse correlation between LAG-3 and IL-17 and IFN-γ expression on CD4 T cells in the spinal cords on day 12 of EAE. *p<0.05 by unpaired two-tailed t test.


A IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice with EAE ( EAE ) co - cultured for 3 days with MOG - specific Tr1 cells ( from mice immunized twice with MOG , RA and IL - 2 and amplified in vitro with MOG , RA and IL - 2 for 8 days ) or control T cells from LNs of mice injected with PBS + DMSO ( Veh ) . Bars are mean + SD of one experiment . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by unpaired two - tailed t test .

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A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test.


B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG - specific Tr1 cells amplified in vitro with MOG , RA and IL - 2 . Results are mean + SEM ( n = 4 or 6 ) . * p < 0 . 05 and * * p < 0 . 01 by two - way ANOVA with Bonferroni post hoc test .

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B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test.


C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells . D Mean absolute numbers of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Results are mean + SEM ( n = 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

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C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells. D Mean absolute numbers of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Results are mean + SEM (n=6). *p<0.05 by unpaired two-tailed t test.


C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells.


E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + . F Mean percentages of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Results are mean + SEM ( n = 6 ) . * * p < 0 . 01 by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+. F Mean percentages of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Results are mean + SEM (n=6). **p<0.01 by unpaired two-tailed t test.


E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + .

E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+.


G EAE clinical scores of WT and IL - 10 - / - mice immunized s . c . with MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 6 or 7 ) . * * * p < 0 . 001 , WT MOG + IL - 2 + RA vs WT Veh , # # p < 0 . 01 , # # # p < 0 . 001 , IL - 10 - / - MOG + IL - 2 + RA vs IL - 10 - / - Veh by two - way ANOVA and Tukey post hoc test .

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G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test.


Retinal inflammatory scores of mice immunized s . c . with PBS + DMSO ( Veh ) , IRBP + DMSO ( IRBP ) or IRBP + RA + IL - 2 21 and 7 days before induction of EAU by s . c . injection of IRBP emulsified in CFA . Bars are mean + SEM ( n = 6 ) , * p < 0 . 05 and * * * p < 0 . 001 , IRBP + IL - 2 + RA vs Veh , # # # p < 0 . 001 IRBP + IL - 2 + RA vs IRBP by two - way ANOVA with Tukey post hoc test .

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Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test.


Representative fundus images of each group of mice 35 days after induction of EAU . Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze , where the image appears blurred .

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Representative fundus images of each group of mice 35 days after induction of EAU. Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze, where the image appears blurred.


Retinal inflammation scores in HEL - TCR transgenic mice were s . c . injected with PBS + DMSO ( Veh ) or HEL + RA + IL - 2 on pn 18 ( 1x ) or on pn 18 and 25 ( 2x ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * p < 0 . 05 and * * * p < 0 . 001 , HEL + IL - 2 + RA 1x vs Veh , # # p < 0 . 01 HEL + IL - 2 + RA 2x vs Veh by two - way ANOVA with Tukey post hoc test .

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Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test.


Representative photographs of each group of retinal fundus of HEL - TCR transgenic mice at pn 35 . Arrowheads indicate representative areas with retinal vasculitis .

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Representative photographs of each group of retinal fundus of HEL-TCR transgenic mice at pn 35. Arrowheads indicate representative areas with retinal vasculitis.


E Representative FACS plots of IL - 17 + CD4 or IL - 10 + CD4 T cells in the retina on day 36 of HEL - TCR transgenic mice . Cells were gated on live single CD3 + CD4 + and CD3 + CD4 + LAG - 3 + Foxp3 - respectively .

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E Representative FACS plots of IL-17+ CD4 or IL-10+ CD4 T cells in the retina on day 36 of HEL-TCR transgenic mice. Cells were gated on live single CD3+CD4+ and CD3+CD4+LAG-3+Foxp3- respectively.


F Mean absolute number of IL - 17 + CD4 T cells in the retina ( upper panels ) or mean percentages of IL - 10 + LAG - 3 + Foxp3 - CD4 T cells in the spleens of of HEL - TCR transgenic mice with vasculitis ( lower panels ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * * p < 0 . 01 and * * * p < 0 . 001 by one way ANOVA with Bonferroni post hoc test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Mean absolute number of IL-17+ CD4 T cells in the retina (upper panels) or mean percentages of IL-10+ LAG-3+ Foxp3- CD4 T cells in the spleens of of HEL-TCR transgenic mice with vasculitis (lower panels). Results are mean + SEM (n=3, 4 or 5). **p <0.01 and ***p <0.001 by one way ANOVA with Bonferroni post hoc test.


A C57BL / 6 WT or Rag2 − / − mice were immunosuppressed with hydrocortisone + / − FK506 and infected with 105 AF CEA10 conidia intranasally . Mortality was assessed using a 20 % weight loss endpoint . Mortality was significantly increased in FK506 immunosuppressed mice reaching 100 % by day 10 p . i . ( P = 0 . 003 for HC versus HC + FK506 in Rag2 − / − using log - rank test , n = 10 per group ) .

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A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group).


B , C PAS - stained lung sections of Rag2 − / − mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ . FK506 - treated animals had increased inflammation at day 2 p . i . compared to control . P = 0 . 0126 ( using Student ' s t - test ) , n = 3 - 5 per group . ( C ) Close - up of an infectious focus showing increased inflammation and fungal growth in the FK506 - treated group . Scale bar : 400 μm .

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B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm.


D , E Murine macrophages ( J774A . 1 ) were pre - treated with hydrocortisone , and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy . The nuclei are indicated in red , and the GR is indicated in green . Scale bar : 50 μm . FK506 did not cause increased translocation of the glucocorticoid receptor . Data represent mean + SEM calculated from 10 fields of vision . One out of 2 experiments is shown .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E Murine macrophages (J774A.1) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . A Weight was monitored daily for 7 days p . i . Immunosuppressed mice showed increased weight loss through days 1 - 3 p . i . Data points represent mean ± SEM .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1-3 p.i. Data points represent mean ± SEM.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . B BALs were performed over a 3 - day period p . i . and fungal killing assessed by enumerating CFUs . Fungal killing was significantly impaired in immunosuppressed mice . Error bars indicate SEM .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . C , D Flow cytometry analysis of the CD45 + leucocyte populations in BAL . Representative dot plots for CD45 + cells ( C ) and percentages of Ly - 6G - positive neutrophils infiltrating the infected lungs over a 48 - h p . i . time course ( D ) . The bars indicate the mean percentage ( ± SEM ) of neutrophils of total CD45 + leucocytes ( n = 5 mice per group ) . Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p . i .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n. C, D Flow cytometry analysis of the CD45+leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . E PAS - stained lung sections of immunocompetent and FK506 - treated animals at 6 h p . i . Scale bar : 50 μm . IC animals showed inflammatory cell infiltrates around conidia ( black arrows ) , whereas in FK506 - treated animals , only uptake of conidia by the resident macrophage population could be seen .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ . Bars represent mean percentages ( ± SEM ) , n = 3 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . G Luminex multiplex cytokine analysis of BAL SNs at 6 h p . i . Bars represent mean ± SEM .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . A , B Absolute neutrophil numbers in treated and untreated mpx : GFPlarvae were quantified by direct counting of fluorescent neutrophils in non - infected whole fish ( n = 11 - 13 per group ) . There were no significant differences .

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At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.A, B Absolute neutrophil numbers in treated and untreated mpx:GFPlarvae were quantified by direct counting of fluorescent neutrophils in non-infected whole fish (n = 11-13 per group). There were no significant differences.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . C For survival analysis , lyz : dsRedlarvae were microinjected with ˜ 10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan - Meier analysis for survival over 6 days post - infection ( n = 16 per group ) . Immunosuppression led to a significant increase in mortality .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.C For survival analysis, lyz:dsRedlarvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan-Meier analysis for survival over 6 days post-infection (n = 16 per group). Immunosuppression led to a significant increase in mortality.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . D TNF - α mRNA levels were measured in whole non - infected or infected larvae 48 h p . i . Immunosuppression led to significant and almost complete inhibition of TNF - α expression during infection .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. D TNF-α mRNA levels were measured in whole non-infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF-α expression during infection.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFP larvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . E - G Lyz : dsRedlarvae were infected with ˜ 50 eGFP - expressing conidia of A . fumigatus and were imaged daily from 24 h p . i . ( E ) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ . A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown ( 60 h p . i . ) . The arrows indicate infectious foci . ( F ) Neutrophil recruitment was minimal throughout the first 60 h p . i . in immunosuppressed larvae ( n = 8 per group ) . ( G ) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. E-G Lyz:dsRedlarvae were infected with ˜50 eGFP-expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group).


A J774A . 1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . FK506 significantly reduced TNF - α levels at 5 and 10 ng / ml for AF . A significant reduction in TNF - α was also seen for zymosan , but not LPS .

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A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS.


B J774A . 1 cells were treated with FK506 ( 10 ng / ml ) and either left non - stimulated ( NS ) or stimulated with AF SC ( MOI = 1 ) for 6 h . TNF - α mRNA levels were quantified from whole - cell RNA extracts by RT - PCR . FK506 significantly reduced TNF - α expression .

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B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT-PCR. FK506 significantly reduced TNF-α expression.


C J774A . 1 macrophages were treated with control ( Ctrl . ) or calcineurin A ( CnA ) - targeting siRNA ( 50 nM ) and stimulated with either AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . CnA siRNA led to significant reductions in both AF - and zymosan - dependent TNF - α levels , but had no significant effect on LPS responses .

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C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.


A J774A . 1macrophages were stimulated with live AF SC ( MOI = 5 ) , and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2 . NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p . i .

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A J774A.1macrophages were stimulated with live AF SC (MOI=5), and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2. NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p.i.


B J774A . 1 macrophages were stimulated with live or fixed swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFAT translocation was assessed by confocal microscopy . NFAT is indicated in green , the nuclei are indicated in red , and AF conidia can be seen in pink . Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan . Data represent mean ± SEM . Scale bar : 50 μm . Arrows indicate A . fumigatus or zymosan - positive cells .

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B J774A.1 macrophages were stimulated with live or fixed swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFAT translocation was assessed by confocal microscopy. NFAT is indicated in green, the nuclei are indicated in red, and AF conidia can be seen in pink. Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan. Data represent mean ± SEM. Scale bar: 50 μm. Arrows indicate A. fumigatus or zymosan-positive cells.


C J774A . 1 macrophages were stimulated with zymosan after FK506 ( 10 ng / ml ) or control treatment , and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF - κB translocation . NFATc2 and NF - κB nuclear translocation was observed , which was maximal at 1 h p . i . , in stimulated macrophages . FK506 specifically inhibited NFATc2 translocation , but had no effect on NF - κB .

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C J774A.1 macrophages were stimulated with zymosan after FK506 (10 ng/ml) or control treatment, and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF-κB translocation. NFATc2 and NF-κB nuclear translocation was observed, which was maximal at 1 h p.i., in stimulated macrophages. FK506 specifically inhibited NFATc2 translocation, but had no effect on NF-κB.


D J774A . 1 macrophages were pre - treated with FK506 ( 10 ng / ml ) or vehicle and stimulated with live swollen conidia or zymosan . NFATc2 translocation was evaluated 30 min after stimulation . Box and whisker plot shows median ± maximum / minimum . Scale bar : 50 μm . FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan . Statistical analysis was carried out by Student ' s t - test . NS , non - stimulated . Arrows indicate A . fumigatus or zymosan - positive cells .

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D J774A.1 macrophages were pre-treated with FK506 (10 ng/ml) or vehicle and stimulated with live swollen conidia or zymosan. NFATc2 translocation was evaluated 30 min after stimulation. Box and whisker plot shows median ± maximum/minimum. Scale bar: 50 μm. FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan. Statistical analysis was carried out by Student's t-test. NS, non-stimulated. Arrows indicate A. fumigatus or zymosan-positive cells.


A BMDMs from WT , Dectin - 1 − / − or MyD88 − / − mice were infected with swollen conidia ( MOI = 1 ) , and TNF - α was measured in the SN after overnight incubation . TNF - α levels were significantly reduced in Dectin1 − / − and Myd88 − / − BMDMs .

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A BMDMs from WT, Dectin-1−/− or MyD88−/− mice were infected with swollen conidia (MOI = 1), and TNF-α was measured in the SN after overnight incubation. TNF-α levels were significantly reduced in Dectin1−/− and Myd88−/− BMDMs.


B BMDMs were stimulated with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation . Whilst almost complete abrogation of NFAT translocation was seen in Dectin1 − / − macrophages stimulated with zymosan , NFAT responses to AF were unaffected .

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B BMDMs were stimulated with swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation. Whilst almost complete abrogation of NFAT translocation was seen in Dectin1−/−macrophages stimulated with zymosan, NFAT responses to AF were unaffected.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( C ) TNF - α was measured in the SN after overnight incubation . Syk inhibition significantly reduced swollen conidia - dependent or zymosan - dependent TNF - α levels .

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C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (C) TNF-α was measured in the SN after overnight incubation. Syk inhibition significantly reduced swollen conidia-dependent or zymosan-dependent TNF-α levels.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( D ) NFATc2 translocation was quantified by confocal microscopy . Syk inhibition impaired zymosan - dependent NFAT translocation , but did not affect AF - dependent NFAT translocation .

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C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (D) NFATc2 translocation was quantified by confocal microscopy. Syk inhibition impaired zymosan-dependent NFAT translocation, but did not affect AF-dependent NFAT translocation.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( E ) Whole - cell lysates of Syk or Ctrl . siRNA - treated cells were separated by SDS - PAGE , followed by Western blotting , and membranes were probed with anti - Syk and anti - β - actin antibodies .

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C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (E) Whole-cell lysates of Syk or Ctrl. siRNA-treated cells were separated by SDS-PAGE, followed by Western blotting, and membranes were probed with anti-Syk and anti-β-actin antibodies.


F J774A . 1 macrophages were pre - treated with piceatannol and infected with swollen conidia ( MOI = 5 ) . Phagocytosis was quantified by FACS 30 min after stimulation . Syk inhibition led to impaired phagocytosis of zymosan , but had no effect on phagocytosis of SC .

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F J774A.1 macrophages were pre-treated with piceatannol and infected with swollen conidia (MOI = 5). Phagocytosis was quantified by FACS 30 min after stimulation. Syk inhibition led to impaired phagocytosis of zymosan, but had no effect on phagocytosis of SC.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( G ) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (G) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( H ) For quantification of NF - κB translocation , nuclear extracts were separated by SDS - PAGE , followed by Western blotting . Membranes were probed with anti - NF - κB p65 and anti - HDAC1 antibodies .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (H) For quantification of NF-κB translocation, nuclear extracts were separated by SDS-PAGE, followed by Western blotting. Membranes were probed with anti-NF-κB p65 and anti-HDAC1 antibodies.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( I ) After overnight stimulation , TNF - α was measured in the SN by ELISA . Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF , but not ionomycin ( G ) . In addition , inhibition of phagocytosis lead to increased translocation of NF - κB ( H ) and increased levels of TNF - α in response to either zymosan or AF .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (I) After overnight stimulation, TNF-α was measured in the SN by ELISA. Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF, but not ionomycin (G). In addition, inhibition of phagocytosis lead to increased translocation of NF-κB (H) and increased levels of TNF-α in response to either zymosan or AF.


A J774A . 1 macrophages were pre - treated with bafilomycin A1 ( 100 nM ) and stimulated with swollen conidia ( MOI = 3 ) for 30 min . NFATc2 translocation was assessed by confocal microscopy . Acidification was required for NFATc2 translocation .

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A J774A.1 macrophages were pre-treated with bafilomycin A1 (100 nM) and stimulated with swollen conidia (MOI = 3) for 30 min. NFATc2 translocation was assessed by confocal microscopy. Acidification was required for NFATc2 translocation.


B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP - expressing swollen conidia by confocal microscopy . Scale bar : 10 μm . The arrow indicates a TLR9 - positive AF - containing phagosome .

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B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP-expressing swollen conidia by confocal microscopy. Scale bar: 10 μm. The arrow indicates a TLR9-positive AF-containing phagosome.


C BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 1 ) , and after overnight stimulation , TNF - α was measured in the SN by ELISA . TLR9 − / − BMDMs had significantly lower TNF - α levels .

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C BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 1), and after overnight stimulation, TNF-α was measured in the SN by ELISA. TLR9−/− BMDMs had significantly lower TNF-α levels.


D , E BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 5 ) for 30 min , and NFATc2 translocation was quantified by ( D ) confocal microscopy . TLR9 − / − BMDMs exhibited reduced NFATc2 translocation by both approaches .

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D, E BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 5) for 30 min, and NFATc2 translocation was quantified by (D) confocal microscopy. TLR9−/− BMDMs exhibited reduced NFATc2 translocation by both approaches.


G BMDMs from WT or MyD88 − / − mice were pre - treated with ODN2088 ( 10 μM ) and infected with swollen conidia ( MOI = 5 ) for 30 min . NFATc2 translocation was quantified by confocal microscopy . In both , WT and MyD88 − / − BMDMs , blocking TLR9 significantly impaired NFATc2 translocation .

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G BMDMs from WT or MyD88−/−mice were pre-treated with ODN2088 (10 μM) and infected with swollen conidia (MOI = 5) for 30 min. NFATc2 translocation was quantified by confocal microscopy. In both, WT and MyD88−/− BMDMs, blocking TLR9 significantly impaired NFATc2 translocation.


A , B J774A . 1 macrophages were pre - treated with the BTK inhibitor LFM - A13 ( 12 . 5 μM ) and infected with swollen conidia . ( A ) After overnight stimulation , TNF - α was measured in the SN by ELISA .

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A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA.


A , B J774A . 1 macrophages were pre - treated with the BTK inhibitor LFM - A13 ( 12 . 5 μM ) and infected with swollen conidia . ( B ) After 30 min of stimulation , NFATc2 translocation was quantified by confocal microscopy . Both TNF - α levels and NFATc2 translocation were significantly reduced by BTK blockade .

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A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade.


C J774A . 1 macrophages were treated with a control or BTK - targeting siRNA ( 25 nM ) , and NFAT translocation was quantified by confocal microscopy . BTK knock - down led to a significant reduction in NFATc2 translocation in response to AF .

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C J774A.1 macrophages were treated with a control or BTK-targeting siRNA (25 nM), and NFAT translocation was quantified by confocal microscopy. BTK knock-down led to a significant reduction in NFATc2 translocation in response to AF.


D , E J774A . 1 macrophages were treated with a control , BTK ( 25 nM ) - or TLR9 ( 75 nM ) - targeting siRNA and additionally pre - treated with either ODN2088 ( 10 μM ) or the BTK inhibitor LFM - A13 ( 12 . 5 μM ) . The cells were infected with swollen conidia ( MOI = 5 ) , and NFATc2 translocation was measured by confocal microscopy . Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade .

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D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.


Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . Cells were pre - treated for 1 h with FK506 ( 10 ng / ml ) , the NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in the cell culture supernatant by ELISA . A Representative FACS - phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples .

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Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples.


Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . Cells were pre - treated for 1 h with FK506 ( 10 ng / ml ) , the NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in the cell culture supernatant by ELISA . B Inhibition of alveolar macrophage responses to AF with FK506 , MCV1 or SC514 led to a significant reduction in TNF - α levels .

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Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.


B . Survival curves of Bcs1l homozygotes without ( GRAC ) and with ( GROX ) alternative oxidase ( AOX ) expression ( n = 18 - 21 / group ) . The median survival of GRAC mice was 210 days and of GROX mice 589 days with no gender difference Statistics : The survival data was analyzed using Log - rank ( Mantel - Cox ) test ( p < 0 . 0001 )

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B. Survival curves of Bcs1l homozygotes without (GRAC) and with (GROX) alternative oxidase (AOX) expression (n=18-21/group). The median survival of GRAC mice was 210 days and of GROX mice 589 days with no gender difference Statistics: The survival data was analyzed using Log-rank (Mantel-Cox) test (p<0.0001)


C . Weight of mice from P50 to P200 ( n > 10 / group ) and at P600 ( n = 4 - 6 / group ) .

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C. Weight of mice from P50 to P200 (n>10/group) and at P600 (n=4-6/group).


D . Blood lactate to glucose ratio at P200 ( n = 8 / group ) Bar graphs represent mean ± SD The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on

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D. Blood lactate to glucose ratio at P200 (n=8/group) Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Dual energy X - ray absorptiometry ( DEXA ) analysis ( n = 10 / group ) of ( E ) lean mass , ( F ) fat mas at P98 Bar graphs represent mean ± SD . The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on

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Dual energy X-ray absorptiometry (DEXA) analysis (n=10/group) of (E) lean mass, (F) fat mas at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Dual energy X - ray absorptiometry ( DEXA ) analysis ( n = 10 / group ) o ( G ) bone mineral density at P98 Bar graphs represent mean ± SD . The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on

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Dual energy X-ray absorptiometry (DEXA) analysis (n=10/group) o (G) bone mineral density at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Indirect calorimetric measurement ( n = 10 / group ) of ( H ) heat productio at P77 Bar graphs represent mean ± SD The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on

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Indirect calorimetric measurement (n=10/group) of (H) heat productio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Indirect calorimetric measurement ( n = 10 / group ) o ( I ) respiratory exchange ratio at P77 Bar graphs represent mean ± SD The dat were analyzed using Kruskal - Wallis and Mann - Whitney U tests for selected comparison . Significant differences between groups ( p value ) are indicated on

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Indirect calorimetric measurement (n=10/group) o (I) respiratory exchange ratio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


A . Hematoxylin - eosin - stained cross sections of heart and kidney at P200 .

A. Hematoxylin-eosin-stained cross sections of heart and kidney at P200.


B . Weight of heart and kidney at P200 ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

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B. Weight of heart and kidney at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


Sirius Red staining ( C ) for fibrosis in liver , heart and kidney at

Sirius Red staining (C) for fibrosis in liver, heart and kidney at P200


( D ) Quantification of fibrosis in myocardium , kidney cortex and liver ( n = 5 - 7 ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

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(D) Quantification of fibrosis in myocardium, kidney cortex and liver (n=5-7) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


E - F . Echocardiography data ( n = 4 - 6 / group ) showing ( E ) ejection fraction ( EF ) and fractional shortening ( FS ) . ( F ) Systolic ( LV Vol ; S ) and diastolic ( LV Vol ; D ) left ventricle volume in mice Bar graphs represent mean ± SD Statistics One - way ANOVA followed by unpaired t - test with Welch " s correction for graph

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E-F. Echocardiography data (n=4-6/group) showing (E) ejection fraction (EF) and fractional shortening (FS). (F) Systolic (LV Vol;S) and diastolic (LV Vol;D) left ventricle volume in mice Bar graphs represent mean±SD Statistics One-way ANOVA followed by unpaired t-test with Welch"s correction for graph E


G . Expression of cardiac hypertrophy and fibrosis - associated genes in the pre - symptomatic ( P150 ) heart ( n = 6 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

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G. Expression of cardiac hypertrophy and fibrosis-associated genes in the pre-symptomatic (P150) heart (n=6/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


H , I . 24 h excretion of ( H ) albumin ( I ) creatinine in urine at P200 ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s tes and Mann - Whitney U tests ( for graph I )

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H, I. 24 h excretion of (H) albumin (I) creatinine in urine at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s tes and Mann-Whitney U tests (for graph I)


J , K . Liver enzymes ( J ) alanine aminotransferase ( ALT ) and ( K ) alkaline phosphatase ( ALP ) in plasma at P200 ( n = 8 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

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J, K. Liver enzymes (J) alanine aminotransferase (ALT) and (K) alkaline phosphatase (ALP) in plasma at P200 (n=8/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


A . GFAP staining for cerebral astrocytes , the barrel field of the primary somatosensory cortex ( S1BF ) highlighted with

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A. GFAP staining for cerebral astrocytes, the barrel field of the primary somatosensory cortex (S1BF) highlighted with brackets


B . Mitochondrial ultrastructure in cardiomyocytes , kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy . Scale bar 1 µm .

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B. Mitochondrial ultrastructure in cardiomyocytes, kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy. Scale bar 1 µm.


( C ) Average cross - sectional area of mitochondrio ( n = 3mice / group Bar graphs represent mean ± SD . Statistics for graph One - way ANOVA followed by Tukey "

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(C) Average cross-sectional area of mitochondrio (n=3mice/group Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s


( D number of cristae per mitochondrio ( n = 3mice / group ) Bar graphs represent mean ± SD . Statistics for graph ) : One - way ANOVA followed by Tukey "

(D number of cristae per mitochondrio (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph ): One-way ANOVA followed by Tukey"s


( E ) cristae thickness , and ( F ) average distance between cristae in mitochondria ( n = 3mice / group ) Bar graphs represent mean ± SD . Statistics for graph One - way ANOVA followed by Tukey "

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(E) cristae thickness, and (F) average distance between cristae in mitochondria (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s


A - C . Principal component analysis of transcriptome data from ( A ) heart , ( B ) kidney and ( C ) liver . D - F . Heat map visualization of pathway enrichment analysis ( Reactome database ) . Full pathway analysis is provided in Appendix Table S1 . Benjamin - Hochberg FDR - corrected p values are color - labelled as indicated for the three comparisons .

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A-C. Principal component analysis of transcriptome data from (A) heart, (B) kidney and (C) liver. D-F. Heat map visualization of pathway enrichment analysis (Reactome database). Full pathway analysis is provided in Appendix Table S1. Benjamin-Hochberg FDR-corrected p values are color-labelled as indicated for the three comparisons.


Gene expression of major transcriptional regulators of energy metabolism ( PPAR - α , mitochondrial biogenesis ( PGC - 1α , TFAM ) in heart at P150 . ( n = 6 / group ) Box plot whiskers represent quartiles , maximum and minimum value ( relative fold change ' FC ' to WT ) . Statistics : one - way ANOVA followed by Tukey ' s test .

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Gene expression of major transcriptional regulators of energy metabolism (PPAR-α, mitochondrial biogenesis (PGC-1α, TFAM) in heart at P150. (n=6/group) Box plot whiskers represent quartiles, maximum and minimum value (relative fold change 'FC' to WT). Statistics: one-way ANOVA followed by Tukey's test.


Gene expression of major transcriptional regulators of energy metabolism HIF - 1α ) and stress responses ( ATF4 ) in heart at P150 . ( n = 6 / group ) Box plot whiskers represent quartiles , maximum and minimum value ( relative fold change ' FC ' to WT ) . Statistics : one - way ANOVA followed by Tukey ' s test .

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Gene expression of major transcriptional regulators of energy metabolism HIF-1α) and stress responses (ATF4) in heart at P150. (n=6/group) Box plot whiskers represent quartiles, maximum and minimum value (relative fold change 'FC' to WT). Statistics: one-way ANOVA followed by Tukey's test.


L . Expression of cIII stress signature genes in heart , kidney , and liver . ( n = 6 / group )

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L. Expression of cIII stress signature genes in heart, kidney, and liver. (n=6/group)


A . Significantly altered metabolites ( FDR < 0 . 2 ; P < 0 . 05 , n = 5 / group ) in presymptomatic ( P150 ) heart tissue of GRAC ( red ) and GROX ( green ) mice Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey "

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A. Significantly altered metabolites (FDR<0.2; P<0.05, n=5/group) in presymptomatic (P150) heart tissue of GRAC (red) and GROX (green) mice Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s


B . Significantly altered metabolites ( FDR < 0 . 2 ; P < 0 . 05 , n = 5 / group ) in liver tissue of GRAC ( red ) and GROX ( green ) mice Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey "

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B. Significantly altered metabolites (FDR<0.2; P<0.05, n=5/group) in liver tissue of GRAC (red) and GROX (green) mice Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s


C - F . Concentrations of the TCA cycle intermediates ( C ) fumarate and ( D ) malate , ( E ) adenylate energy charge and ( F ) NADH / NAD + ratio ( n = 5 / group ) Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey "

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C-F. Concentrations of the TCA cycle intermediates (C) fumarate and (D) malate, (E) adenylate energy charge and (F) NADH/NAD+ ratio (n=5/group) Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s


( G ) Proline concentratio in heart tissue at P200 ( # below detection limit Bar graph represent mean ±

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(G) Proline concentratio in heart tissue at P200 (# below detection limit Bar graph represent mean±S


mRNA expression of proline synthesis - related genes ( n = 6 / group ) ) ( H ) Aldh18a1 and ( I ) Pycr1 at P150 box plots of mRNA expressions represent quartiles , minimum , and maximum value ( relative fold change " FC & qu Statistics for graph one - way ANOVA followed by Tukey "

mRNA expression of proline synthesis-related genes (n=6/group)) (H) Aldh18a1 and (I) Pycr1 at P150 box plots of mRNA expressions represent quartiles, minimum, and maximum value (relative fold change "FC&qu Statistics for graph one-way ANOVA followed by Tukey"s


A . Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes ( SCs )

A. Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes (SCs)


B - C . RISP per CORE1 ratio in ( B ) SC1 and ( C ) free cIII2 .

B-C. RISP per CORE1 ratio in (B) SC1 and (C) free cIII2.


Spectrophotometric assay of ( D ) cI , ( E ) cII activity in isolated mitochondria at P200 ( n = 4 - 6 / group ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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Spectrophotometric assay of (D) cI, (E) cII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


Spectrophotometric assay o ( F ) cIII activity in isolated mitochondria at P200 ( n = 4 - 6 / group ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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Spectrophotometric assay o (F) cIII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


G . CIV activity as measured using an oxygraph Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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G. CIV activity as measured using an oxygraph Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


H . n - Propyl gallate - sensitive AOX mediated cI and cII - linked state 3 respiration ( n = 7 / group , Mann - Whitney U test ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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H. n-Propyl gallate-sensitive AOX mediated cI and cII-linked state 3 respiration (n=7/group, Mann-Whitney U test) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


CI - linked ( I state 3 respiration in isolated heart , kidney and liver mitochondria ( n = 8 / group ) We used malate , pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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CI-linked (I state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) We used malate, pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


cI - and cII - linke state 3 respiration in isolated heart , kidney and liver mitochondria ( n = 8 / group ) we added the CII substrate , succinate , to obtain CI & CII - linked respiration in presence of saturating ADP . Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s test . Significant differences between groups ( p value ) are indicated on

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cI- and cII-linke state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) we added the CII substrate, succinate, to obtain CI&CII-linked respiration in presence of saturating ADP. Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


A . Amplex Red - based detection of H2O2 emission , a surrogate for respiratory chain - derived ROS , during cI and cII - linked state 3 respiration ( n = 6 / group ) Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey "

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A. Amplex Red-based detection of H2O2 emission, a surrogate for respiratory chain-derived ROS, during cI and cII-linked state 3 respiration (n=6/group) Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"


B. Gene expression related to ROS scavenging


C . Total glutathion in heart and liver tissue ( n = 5 / group Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey "

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C. Total glutathion in heart and liver tissue (n=5/group Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"


D . Urinary isoprostanes per creatinine as a measure of oxidative stress ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : Mann - Whitney U test ( graph

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D. Urinary isoprostanes per creatinine as a measure of oxidative stress (n=4/group) Bar graphs represent mean±SD Statistics: Mann-Whitney U test (graph D


E and F . Representative images ( D ) of tissue sections immunostained with an antibody against 4 - hydroxynonenal ( 4 - HNE ) , a lipid peroxidation marker . ( E ) Quantification of the 4 - HNE immunostaining .

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E and F. Representative images (D) of tissue sections immunostained with an antibody against 4-hydroxynonenal (4-HNE), a lipid peroxidation marker. (E) Quantification of the 4-HNE immunostaining.


A . Heatmap visualization of the expressions of selected nitric oxide ( NO ) - related genes .

A. Heatmap visualization of the expressions of selected nitric oxide (NO)-related genes.


B - E . Gene expression of Ca2 + channel gene ( B ) ryanodin receptor , RYR2 , ( C ) sarco ( endo ) plasmic reticulum Ca2 + - ATPase ( SERCA2 or ATP2A2 ) , ( D ) phospholamban ( PLN ) , and ( E ) endothelial nitric oxide synthase NOS3 ( n = 6 / group ) Box plots for mRNA expressio represent quartiles , minimum and maximum value ( relative fold change " FC & qu Statistics : one - way ANOVA followed by Tukey " s test for Significant differences between groups ( p value ) are indicated on

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B-E. Gene expression of Ca2+ channel gene (B) ryanodin receptor, RYR2, (C) sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2 or ATP2A2), (D) phospholamban (PLN), and (E) endothelial nitric oxide synthase NOS3 (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs


Expression of neuronal nitric oxide synthase ( NOS1 ) mRNA ( F Bar graph represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s test for Significant differences between groups ( p value ) are indicated on

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Expression of neuronal nitric oxide synthase (NOS1) mRNA (F Bar graph represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs


Expression of neuronal nitric oxide synthase ( NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane ( G )

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Expression of neuronal nitric oxide synthase (NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane (G)


H . Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 ( DDAH1 ) ( n = 6 / group ) Box plots for mRNA expressio represent quartiles , minimum and maximum value ( relative fold change " FC & qu Statistics : one - way ANOVA followed by Tukey " s test for Significant differences between groups ( p value ) are indicated on

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H. Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs


I . Cardia citrulline to arginine ratio ( n = 5 / group ) Bar graph represent mean ±

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I. Cardia citrulline to arginine ratio (n=5/group) Bar graph represent mean±SD


J . Cardiac protein nitrotyrosine quantified from Western

J. Cardiac protein nitrotyrosine quantified from Western blots


K . Nitrite concentration in heart tissue ( n = 4 / group ) Bar graph represent mean ±

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K. Nitrite concentration in heart tissue (n=4/group) Bar graph represent mean±SD


( A ) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress ( 200μM H2O2 ) . Biological triplicate data is depicted at each time point relative to control cells .

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(A) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress (200μM H2O2). Biological triplicate data is depicted at each time point relative to control cells.


( B ) MCF10A cells were exposed to oxidative stress ( 200μM H2O2 ) and processed for small RNA - sequencing . The log2 - fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted .

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(B) MCF10A cells were exposed to oxidative stress (200μM H2O2) and processed for small RNA-sequencing. The log2-fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted.


( A ) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A cells in response to oxidative stress . A single probe complementary to pre - tRNATyrGUA , mature tRNATyrGUA , and tRFTyrGUA expression was 32P - labeled and used for detection . ( B ) Quantification of pre - tRNATyrGUA ( left ) and mature tRNATyrGUA levels ( right ) by northern blot analysis from multiple independent experiments ( normalized to U6 levels ) are shown ( n = 6 ) .

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(A) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A cells in response to oxidative stress. A single probe complementary to pre-tRNATyrGUA, mature tRNATyrGUA, and tRFTyrGUA expression was 32P-labeled and used for detection. (B) Quantification of pre-tRNATyrGUA (left) and mature tRNATyrGUA levels (right) by northern blot analysis from multiple independent experiments (normalized to U6 levels) are shown (n=6).


( C ) MCF10A cells were exposed to oxidative stress ( 200μM H2O2 ) once daily for five continuous days . ( D ) Quantification of mature tRNATyrGUA bands by northern blot after cells were treated once daily for five continuous days ( normalized to U6 ) from multiple independent experiments ( n = 12 ) .

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(C) MCF10A cells were exposed to oxidative stress (200μM H2O2) once daily for five continuous days. (D) Quantification of mature tRNATyrGUA bands by northern blot after cells were treated once daily for five continuous days (normalized to U6) from multiple independent experiments (n=12).