Nature | Vol 577 | 30 January 2020 | 683cells during M. tuberculosis infection^5 ; TH1 cells produce interferon
(IFN)γ; and IFNγ inhibits the accumulation of pathogenic neutro-
phils in infected lungs during M. tuberculosis infection^6 ,^7. Hence, the
reduced immune-cell infiltration that we observed in the lungs of
H37RvΔRv0222-infected mice could be due to a higher level of IFNγ-
producing TH1 cells. The bacterial burden in the lung tissue of mice
infected with H37Rv(ΔRv0222 + Rv0222) was much higher than in mice
infected with H37Rv(ΔRv0222 + GFP) (Fig. 1i and Extended Data Fig. 1j).
Together, these results suggest that Rv0222 is an essential virulence
factor for M. tuberculosis.Rv0222 inhibits TRAF
Various pattern-recognition receptors (PRRs) in hosts regulate M. tuber-
culosis-induced cytokine expression by activating mitogen-activated
protein (MAP) kinase and NF-κB pathways. We found that, in the absence
of Rv0222, macrophages infected with H37Rv exhibited much stronger
activation of MAP kinase and NF-κB (Extended Data Fig. 2a), suggesting
that Rv0222 may inhibit the activation of pathways that comprise the p
MAP kinases, the c-Jun amino-terminal kinases ( JNKs, which are down-
stream of MAP kinases) or NF-κB in response to M. tuberculosis infection.
Inhibition of p38, JNK or NF-κB attenuated the enhanced expression levelslog 2 [ΔRv0222/H37Rv]
(MOI = 5, 4 h)a12H37Rv ΔRv051015Fold change inIl1bmRNAP < 0.b c
P < 0.H37Rv ΔRv0204060Fold change inIlmRNAH37Rv ΔRv020406080Fold change inIl12 pmRNAP < 0.d0360255075100125150175Fold change inIl1bmRNA P = 0.P < 0.H37Rv H37Rv + GFP ΔRv0222 + GFP ΔRv0222 + RvP < 0.P < 0.P = 0.P = 0.h0360100200300400Fold change inIlmRNAP = 0.P = 0.P < 0.P = 0.P = 0.e f P = 0.i173002468log[CFU] 10P < 0.P < 0.H37Rv ΔRv0222 + GFP
ΔRv0222 + Rv036010203040Fold change inIl12 pmRNAP = 0.P < 0.P < 0.P = 0.P = 0.P = 0.g
ΔRv
+ RvΔRv
+ GFP× 40× 100H37RvIl1rn 0.68 0.
Tnfsf13b 0.63 0.
Il1rl1 0.54 0.
Il1b 0.49 0.
Il1r1 0.42 0.
Tnfrsf12a 0.32 0.
Tnfrsf23 0.27 0.
Tnfrsf9 0.25 0.
Tgfb2 0.23 0.
Tnfrsf4 0.23 0.
Il1rl2 0.22 0.
Il1a 0.17 0.
Tnfrsf18 0.16 0.
Tnfrsf21 0.14 0.
Tnfrsf8 0.14 0.
Il1f9 0.14 0.
Tnfrsf26 0.14 0.
Il6 0.14 0.
Tnfrsf11b 0.10 0.
Il12a 0.00 0.
Il10ra -0.16 -0.
Il10 -0.28 -0.
–1 0 1
Time (h) Time (h)Time (h) Time (days)Fig. 1 | Rv0222 inhibits host inf lammatory responses. a, Heat map of
downregulated (blue) and upregulated (red) mRNAs from peritoneal
macrophages infected with H37Rv (n = 2) versus ΔRv0222 (n = 2) (multiplicity of
infection (MOI) = 5) for 4 h. Numbers 1 and 2 refer to two independent
experimental analyses of log2(∆Rv0222/H37Rv). b–d, Quantitative
polymerase chain reaction (qPCR) analysis of Il1b (b), Il6 (c) and Il12 p40 (d)
mRNA from the lungs of C57BL/6 mice aerosol-infected with roughly 200
colony-forming units (CFUs) per mouse of H37Rv or ΔRv0222 for 7 days. The
graphs show cumulative data from three independent experiments
(mean ± s.e.m. of n = 18), with red, blue and yellow circles denoting separate
experiments. e–g, qPCR analysis of Il1b (e), Il6 (f) and Il12p40 (g) mRNA
from peritoneal macrophages infected with H37Rv, H37Rv + GFP,
H37Rv(ΔRv0222 + GFP) or H37Rv(ΔRv0222 + Rv0222) for 3 h or 6 h (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. Bar charts show means. h, i, C57BL/6 mice were aerosol-infected
with roughly 200 CFUs per mouse of H37Rv, H37Rv(ΔRv0222 + GFP) or
H37Rv(ΔRv0222 + Rv0222). Histopathology was assessed in lung sections
stained with haematoxylin and eosin from mice infected for 30 days
(h; representative of one experiment with at least three independent
replicates; scale bars, 1,000 μm (top) and 200 μm (bottom)), and bacterial
titres were assessed in lungs from mice infected for 1, 7 or 30 days (i; cumulative
data from three independent experiments; mean ± s.e.m. of n = 9 mice infected
for 1 day and n = 18 mice infected for 7 or 30 days). Two-tailed
unpaired Student’s t-test (b–g) and two-sided Mann–Whitney U-test (i) were
used for statistical analysis. For gel source data, see Supplementary Fig. 1.