Science - USA (2022-02-25)

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can be adoptively transferred and can operate
in a T cell–independent fashion during path-
ogen rechallenge.
Although antigen specificity has long been
considered as a cardinal feature of adaptive


B and T cell memory ( 23 ), innate populations
such as myeloid cells ( 24 ), natural killer (NK)
cells ( 25 , 26 ), and ILC2s ( 27 )havebeenshown
to have“memory-like”properties. To investi-
gate pathogen-specific properties of Tr-ILC3s,

we analyzed whetherC. rodentium–activated
Tr-ILC3s could protect againstListeria
monocytogenes.L. monocytogenesinfection
(LM) elicits IL-23 release that can trigger ILC3
activation and IL-22 production, which is

862 25 FEBRUARY 2022•VOL 375 ISSUE 6583 science.orgSCIENCE


A RorcGFP Il22TdT

CR Abx

D0 D29

CR

D-1 D3 D33

C CR CRACRACR

BCD49a+ ILC3

CCR6+ ILC3

D

G


  • AA/R/O


0

20

40

60

80

IL-22

+ ILC3 (%)

Tr-ILC3

ILC3
Tr-ILC3

ILC3

-2

2
0

CCR6+ ILC3

CD49a+ ILC3

Glycolysis TCA cycle

Oxphos

CCR6+ ILC3

CD49a+ ILC3

CCR6+ ILC3

CD49a+ ILC3

-2

2
0

NES (p<0.05, FDR 25%)

1.0 1.2 1.4

DNA replication

Focal adhesion

Adherens junction

Proteasome

Chemokine signaling

Cytokine/receptor interaction

NK cell mediated cytotoxicity

Cell Cycle

Pathogenic E.Coli infection

Oxidative phosphorylation

Fatty acid metabolism

TCA cycle

Glycolysis

Arg and Pro Metabolism

1.0 1.2

C

E F

1.4

H

0 20 40 60 80 100

0

2

4

6

8

10

Time (min)
I

Tr-ILC3

ILC3

CCR6+ ILC3 CD49a+ ILC3 J
Tr-ILC3

ILC3

0 20 40 60 80 100

0

2

4

6

8

10

Time (min)

OCR (pmol/min)

Oligo FCCP

C
CR
CRA
CRACR

C
CR
CRA
CRACR

R/A

ECAR (mpH/min)

Glucose Oligo 2-DG

0

2

4

6

8

10

Max. glycolysis (mpH/min)
Basal Max

0

2

4

6

8

10

Respiration (pmole/min)


  • 2DG/B/E2DG/B/E/N
    Ulk4
    Rec


114
Sertad1
PcnaNap1l1Ccnyl1Dynll1NaspEgr1FosbIer5Klf4

3

104

105

Sequence reads (normalized)
Ulk4
Rec

114
Sertad1

PcnaNap1l1Ccnyl1Dynll1NaspEgr1FosbIer5Klf4

0

20

40

60

80

100

IL-22

+ ILC3 (%)

Ki-67
CRACR

ILC3
CR
CRACR

0

2

4

6

8

10

12

cells (% of ILC3)

CR CR CRACR

10
Ki67

*
ns
ns

**

+

Ndufa1Ndufa2Ndufa3Ndufa4Ndufa5Ndufa6Ndufa7Ndufa8Ndufa9Ndufa10Ndufa11Ndufa12Ndufab1Ndufb2Ndufb3Ndufb4Ndufb5Ndufb6Ndufb7Ndufb8Ndufb9Ndufb10Ndufb11Ndufc1Ndufc2Ndufs1Ndufs2Ndufs3Ndufs4Ndufs5Ndufs6Ndufs7Ndufs8Ndufv1Ndufv2Ndufv3SdhaSdhbSdhcSdhdUqcrbUqcrc1Uqcrc2Uqcrfs1UqcrqUqcr10UqcrhUqcr11UqcrbCox7bCox7a2Cox8aCox6b1Cox6a1Cox6cAtp5hAtp5j2Atp5kAtp5f1Atp5dAtp5eAtp5g3

Slc2a3PfkpSlc2a1PdhbHk1Gpi1PkmPfklPdha1Hk2AldoaDldAdh5Pfkfb3DlatG6pc3Eno1Akr1a1Pck2LdhaAldh9a1Aldh3a2Pgm1Aldh2LdhbGalm Acsl1Acsl3Acsl4Acsl5Aco2Idh2Idh3aIdh3bIdh3gOgdhSuclg1SdhaSdhbSdhcSdhdFh1Mdh1Mdh2

2.1 7.2

***

Fig. 4. Gene expression analysis and metabolic profiles of Tr-ILC3s.(A) CCR6+
and CD49a+ILC3 from naïve (C) and infected mice (CR, CRA, and CRACR) were
sorted for RNA-seq analysis. (BandC) A heatmap showing the relative expression
levels of differentially expressed genes is shown in (B). In (C), pathway analysis
was performed and gene pathways were organized into clusters to compare CR,
CRA, and CRACR conditions to C (n= 3). FDR, false discovery rate; NES, normalized
enrichment score. (D) Heatmap with clustering of differentially expressed
metabolism-associated genes (n= 3). (EandF) Seahorse analysis of freshly
sorted ILC3s (C) and Tr-ILC3s (CRACR) in response to a mitochondrial uncoupler
[fluoro-carbonylcyanide phenylhydrazone (FCCP)], oligomycin (oligo), rotenone and
antimycin (R/A), and 2-DG. Representative metabolic profiles (OCR and ECAR),


glycolysis, and respiration were determined (three independent experiments;n=3to
4). (G) Effect of OXPHOS inhibition (18 hours) on IL-22 production by intestinal
ILC3s (n= 5). AA, antimycin; R, rotenone; O, oligomycin. (H) Effect of metabolic
pathway inhibition (2 hours) on IL-22 production by intestinal ILC3s (two
independent experiments;n= 3 to 8). B, BPTES; E, etomoxir; N, Nor-NOHA.
(I) Differentially expressed genes in CR and CRACR ILC3s (n= 3). (J) Frequency
of Ki67+ILC3 from infected (CR) and reinfected (CRACR) mice (two independent
experiments;n= 5). Each graph corresponds to the mean ± SEM of the
values obtained. ns indicates not significant, *P< 0.05, **P< 0.01, and ***P<
0.001 using two-tailed unpaired StudentÕsttest [(E) and (F)], two-tailed Mann-
Whitney test [(G) and (J)], or Krustal-Wallis test (H).

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