Nature 2020 01 30 Part.01

686 | Nature | Vol 577 | 30 January 2020

Article

0

50

100

150

Fold change inIl12 p40

mRNA

P = 0.0037

P = 0.0014

P = 0.0009

130

0

2

4

6

8

log

[CFU] 10

P< 0.0001P< 0.0001

P< 0.0001

Untreated

0

20

40

60

80

Fold change inIl12 p40

mRNA

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

Untreated 1714

0

5

10

15

Fold change in

Il1b

mRNA

H37Rv ΔRv0222 + GFP ΔRv0222 + Rv0222 ΔRv0222 + Rv0222(K76A)

P = 0.0022

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

ΔRv0222 + Rv0222 ΔRv0222 + Rv0222(K76A)

× 40

× 100

× 400

×1,000

f

Untreated

0

20

40

60

Fold change in

Il6

mRNA

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

h

i

g

d e

0

100

200

300

Fold change in

Il6

mRNA

P = 0.0065

P = 0.0061

P = 0.0064

b c

0

100

200

300

Fold change in

Il1b

mRNA

ΔRv0222

P = 0.0051

P = 0.0033

P = 0.0019

a

H37Rv

• GFP
  Rv0222
  Rv0222(K76A) Rv0222(K76A) Rv0222(K76A)
  
  
  • H37RvGFPRv0222
  
  
  
  

ΔRv0222

• H37RvGFPRv0222

ΔRv0222

Time (days)

1714

Time (days)

1714

Time (days)

Time (days)

ΔRv0222 + Rv0222 ΔRv0222 + Rv0222(K76A)

Fig. 4 | K11-linked ubiquitination of Rv0222 by ANAPC2 suppresses anti-
tuberculosis immunity. a–c, qPCR analysis of Il1b (a), Il6 (b) and Il12 p40 (c)
mRNA from peritoneal macrophages infected with H37Rv, H37Rv(ΔRv0222 +
GFP), H37Rv(ΔRv0222 + Rv0222) or H37Rv(ΔRv0222 + Rv0222(K76A)) for
the indicated times (MOI = 5) (mean ± s.e.m.). Data are representative of
one experiment with at least three independent biological replicates;
each circle represents one technical repeat. d–i, Six-week-old female
C57BL/6 mice were aerosol-infected with roughly 200 CFUs per mouse
of H37Rv, H37Rv(ΔRv0222 + GFP), H37Rv(ΔRv0222 + Rv0222) or
H37Rv(ΔRv0222 + Rv0222(K76A)). d–f, qPCR analysis of Il1b (d), Il6 (e) and Il12
p40 (f) mRNA in lungs from mice infected for 0, 1, 7 and 14 days. Cumulative
data from three independent experiments (mean ± s.e.m. of n = 6 mice

untreated, n = 9 mice infected for 1 day and n = 18 mice infected for 7 and 14

days). g–i, For lungs of mice infected for 30 days (g, i) or 1 and 30 days (h), we

assessed: histopathology in sections stained with haematoxylin and eosin

(g; scale bars, 1,000 μm (top) and 200 μm (bottom)); bacterial titres

(h; cumulative data from three independent experiments, mean ± s.e.m. of

n = 6 mice infected for 1 day and n = 18 mice infected for 30 days); and acid-fast

staining (i; scale bars, 100 μm (top) and 20 μm (bottom)). The outlined areas in

the top images are enlarged in the bottom images. Two-tailed unpaired

Student’s t-test (a–f) and two-sided Mann–Whitney U-test (h) were used for

statistical analysis. g, i, Data are representative of one experiment with at least

three independent biological replicates.