Variation and adaptation in innate immune responses

Mihai G. Netea ESCMID eLibrary © by author Introducing BCG in Norrbotten, Sweden, 1927-31

Coverage highest in families with TB

Reduction was in infancy, but TB deaths occur later

This made little sense

”One ESCMID could evidently be tempted to findeLibrary an explanation for this much lower mortality among vaccinated children in the idea that BCG provokes a non-specific immunity...” Carl Naeslund 1932 © by author 2 RCTs of BCG-at-birth in LBW children Infant mortality MRR=0.79 (0.61-1.02) Neonatal MRR=0.52 (0.33-0.82)

ESCMIDPIDJ 2012 eLibraryJID 2011 Reduction in neonatal sepsis and respiratory infections NaeslundNot prevention vindicated! © of TBby => Beneficialauthor NSE of BCG Innate versus specific immunity

Innate immunity: Adaptive immunity: - rapid - needs 10-14 days - effective - a specific activation against a - not-specific, indiscriminate particular microorganism, enhancing - lacks immunological memory the effectivity of the response ESCMID eLibrary- builds immunological memory © by author Memory: the ability of a system to store and recall information on previously encountered characteristics

Holometa- Heterometa- Fishes Amphi- Reptiles Birds Mammals bolous bolous bians Placoderms 5% Insects Innate and Gnathostomes Adaptive 95% -450 My Molluscs Innate Agnathans Echinoderms Annelids Urochordates

-800 My Plants -1 By ESCMID eLibrary5 © by author Increased response to secondary infection

ESCMID eLibrary Netea et al: Cell Host and Microbe© by2011 author Innate immunity-dependent protection in mice

ESCMID eLibrary Quintin et al, Cell H&M, 2012 © by author Does this happen in vivo in humans?

ESCMID eLibrary Kleinnijenhuis et al, PNAS, 2012 © by author BCG enhances monocyte-derived cytokines

ESCMID eLibrary Kleinnijenhuis et al, PNAS, 2012 © by author NOD2 needed for BCG effect

ESCMID eLibrary © by author BCG vaccination in vivo yellow fever vaccine

BCG or placebo Yellow fever

-30 0 3 5 7 14 90

ESCMID eLibrary © by author BCG vaccination in vivo yellow fever vaccine

ESCMID eLibrary Arts et al, Cell Host Microbe, 2018 in press © by author BCG vaccination in vivo yellow fever vaccine

BCG or placebo Yellow fever

-30 0 3 5 7 14 90

ESCMID eLibrary Arts et al, Cell Host Microbe, 2018 © in press by author Trained immunity and in vivo yellow fever vaccine

ESCMID eLibrary Arts et al, Cell Host Microbe, 2018 in press© by author Long-term epigenetic reprogramming in myeloid cells

ESCMID eLibrary Netea et al, Science 2016 © by author Long-term epigenetic reprogramming in myeloid cells

RNA-seq expression Non-coding regulatory RNAs

ATAC-seq Open chromatin (i.e. nucleosome- free regions) can be bound by TFs, which can be identified by motif sequence

ChIP-seq Histone tail modifications determine ‘activity’ by attracting TFs CH3 (we use 5 histone modifications)

CH3 WGBS – whole genome bisulfite sequencing DNA methylation maintains DNA in a ESCMID eLibraryclosed state © by author Long-term epigenetic reprogramming in myeloid cells

ESCMID eLibrary Alvarez-Errico et al, Nature Rev Immunol©2015 by author A Similarity betw een samples - spearman correlation

At least 1 BCG sample BCG v baseline all H 3K27ac > 3SD from baseline mean (p<0.05, FC>1.5)

BCG vaccination induces epigenetic reprogramming

correlation Baseline (-28d) BCG 1 month (0d) 0.5 1 B H 3K27ac (p <0.05 FC>1.5) C 646 peaks (456 up, 190 dow n) H 3K27ac (p <0.05) 3 10 time baseline

)

e

%

r 1 month 0

8

o

.

c 0

9

s

(

-

z 2 −10

C

P −3 −20

−40 −20 0 20

k PC1 (48.3%)

a

e

P

/

s H 3K27ac (p <0.05 FC>1.5)

d

a

e

r 10

) 5

%

8

.

5

( 0

3

C −5

P

−10 −20 −10 0 10 20 ESCMID eLibraryPC1 (46.9%)

D Pathw ay name (PANTHER) P-Value F DR Q-Val Enrichment Regio n Hits Inflammatory Fc epsilon RI B cell activatio n 4 .4 2 E-10 6 .72 E-08 9.6 8 5 14 response signaling Arts et al, Cell HostAp o p to sMicrobe,is signaling p a t2018hw ay 7.77E-07 5 .90E-05 6 .2 76 3 12 AK T1 pathway Ras Pathw ay 4 .19E-06 2 .12 E-04 5 .8 6 97 11 CCR1 AK T1 Axo n guid ance me diate d by sem ap h©o rins by9.74 E-06 3 .70 E-0author4 9.8 7 CXCR2 Inflammatio n mediated by chemo kine and cyto kine signaling 5 .3 4 E-05 1.6 2 E-03 3 .3 96 7 15 GPR6 8 INPP5 D EGF recep to r signaling p athw ay 5 .73 E-05 1.4 5 E-03 3 .785 9 13 TNF AIP6 LCP2 F GF signaling p athw ay 7.85 E-05 1.70E-03 3 .6 6 72 13 TNF RSF 1B MAPK 11 VEGF signaling p athw ay 1.2 1E-04 2 .3 0E-03 4 .902 9 TNIP1 MAPK 14 AOAH Pathw ay name (MSigDB) P-Value F DR Q-Val Enrichment Regio n Hits PIK 3 CD F c ep silo n RI signaling p athw ay 8 .19E-11 1.08 E-07 8 .92 5 4 16 AGER PRK CB APOL3 invo lved in Immune System 8.72 E-10 5 .75 E-07 2 .6 6 1 4 9 PRK CD Leuko cyte transendo thelial migratio n 1.01E-08 2 .6 6 E-06 5 .8 915 17 HDAC4 RAC1 Chemo kine signaling p athw ay 3 .3 3 E-08 7.3 3 E-06 4 .8 115 19 LYZ F c gamma R-mediated p hago cyto sis 4 .6 7E-08 7.70E-06 6 .1095 15 OLR1 RAC2 VEGF signaling p athw ay 1.17E-07 1.4 0E-05 7.5 2 6 4 12 RPS6 K A4 VAV3 A H3K27ac signal (p-adj <0.05, FC>1.5)

GPR20 (+39210) PPFIBP2 (+78775)

Responders (R)

NOD2 (-7625) c

a

s

7

d

2

a

K POU5F1B (+13164) e

r

3

Non-responders H TRIM32 (+258984) (NR) BCG 1mo baseline SKIDA1 (+313461)

PHYKPL (-80059) ARHGEF19 (+6344)

SKIDA1 (+312532) PRAG1 (+158812)

HDAC4 (+81643) PRAG1 (+157898) NEBL (-313681) GRM7 (+747209) ZIC1 (+296632) KLF5 (+276306)

B H3K27ac differential peak near NOD2

STAT3 Non-responder BCG 1mo FOS Responder baseline MonocyteTF binding epigenome (ENCODE) predicts response C

808 H3K27ac dynamic RNA seq of 512 genes regions, p <0.05 near H3K27ac dynamic A regionsH3K27ac signal (p-adj <0.05, FC>1.5) GPR20 (+39210) Biological process PPFIBP2 (+78775)

Responders (R)

NOD2 (-7625) c

a

s

7

d

2

a

K POU5F1B (+13164) e

r

3

Non-responders H TRIM32 (+258984) (NR) BCG 1mo baseline SKIDA1 (+313461)

PHYKPL (-80059) ARHGEF19 (+6344) Cellular SKIDA1 (+312532) PRAG1 (+158812) component HDAC4 (+81643) PRAG1 (+157898) NEBL (-313681) GRM7 (+747209) ZIC1 (+296632) KLF5 (+276306) Responders Non-responders ESCMIDB eLibraryH3K27ac differential peak near NOD2 Arts et al, Cell Host Microbe, 2018 © by author

STAT3 Non-responder BCG 1mo FOS Responder baseline TF binding (ENCODE) C

808 H3K27ac dynamic RNA seq of 512 genes regions, p <0.05 near H3K27ac dynamic regions

Biological process

Cellular component

Responders Non-responders Training vs Tolerance ?

metabolism

ESCMID eLibrarysignaling Saeed et al, Science, 2014 © by author Metabolic status in trained monocytes

ESCMID eLibrary Arts et al, Cell Metabolism 2016 © by author Metabolic status in trained monocytes

ESCMID eLibrary © by author amplification loop ToleranceIGF1R vs Training mevalonate PRRs glucose

Akt G-6-P mevalonate mTOR cholesterol PDH Krebs cycle pyruvate HIF-1α citrate Acetyl-CoA malate

fumarate Lactic acid Histone acetyl transferases KDM5 histone demethylase glutamate ESCMID eLibraryH3K27Ac H3K4me3 glutamine © by authorBekkering et al, Cell 2018 Trained immunity acts on myeloid cell progenitors

ESCMID eLibrary Mitroulis et al, Cell 2018 © by author BCG acts on myeloid cell progenitors

ESCMID eLibrary Kaufmann et al, Cell 2018 © by author BCG trained effects are sustainable in vivo

ESCMID eLibrary Kaufmann et al, Cell 2018 © by author BCG vaccination has effects on human bone marrow

In vivo Immune Memory in Humans - The Study Design

BCG

Day0 Day14 Day90 Follow-up

Experimental Group BCG-vaccinated (n=15) Blood Blood Blood

Control Group Bone Bone Non-Vaccinated (n=5) Marrow Marrow ESCMIDPE T/CeLibraryT Scan PE T/CT Scan de Bree et al, unpublished © by author ESCMID eLibrary © by author Differential Analysis (DESeq2)

D0 vs D90 UP DOWN cMonos PB 227 174 cMonos BM 89 85 GMP 57 99 HSPC 1087 130

GO: Biolog Process

granulocyte activation ALOX5 HBB immune response ATP11A HK3 neutrophil mediated immunity C3AR1 HMOX2 secretion by cell CCR2 HPSE myeloid leukocyte mediated immunity CD36 ITGAX secretion CD68 LGALS3 neutrophil activation involved in immune CFP LILRB3 response CHIT1 LYZ neutrophil degranulation CHRNB4 MNDA neutrophil activation CKAP4 MOSPD2 leukocyte degranulation CLEC5A NFASC Neutrophil counts leukocyte mediated immunity CR1 OSCAR Non-BCG BCG myeloid cell activation involved in immune CRISPLD2 PTAFR response CTSS PTPRJ 6.8 7.6 myeloid leukocyte activation CYBB RAB31 p=0.004 regulated exocytosis DNASE1L S100A12 immune effector process FCER1G S100A8 immune system process FCGR2A S100A9 leuk ocyESCMIDte activation involved in immune eLibraryFCN1 SERPINA1 response FGL2 SIRPB1 cell activation involved in immune response FPR1 TARM1 leukocyte activation FPR2 TLR2 exocytosis FRK TNFRSF1B © by authorTTR Conclusions BCG vaccination

• BCG vaccination leads to non-specific protection against unrelated infections: e.g. yellow fever vaccine viremia, malaria

• BCG vaccination induces trained immunity in circulating monocytes

• BCG induces long-term reprogramming at the level of myeloid cell progenitors in the bone marrow in mice and humans • Responses ESCMID to other vaccines eLibrary are influenced as well © by author ESCMID eLibrary

Netea et al, Science 2016 © by author Thank you !

Department of Internal Medicine - Radboud Bonn University - LIMES Rob Arts Katarzyna Placek Bas Blok Anette Christ Simone Moorlag Eicke Latz Charlotte de Bree Andreas Schlitzer James Cheng Joachim Schultze Siroon Bekkering Jessica Quintin Harvard University Johanneke Kleinnijenhuis Ramnik Xavier Marije Oosting Jos W.M. van der Meer University of Minho, Braga Niels Riksen Agostinho Carvalho Reinout van Crevel Ricardo Silvestre Leo Joosten Fernando Rodrigues Dept. Molecular Biology - Radboud University of Melbourne Boris Novakovic Nicole Messina Sadia Saeed Nigel Curtis Joost Martens Athens University Colin Logie Evangelos Giamarellos Henk Stunnenberg Dresden University East Tennessee State University Ioannis Mitroulis David Williams ESCMIDTriantafyllos Chavakis eLibrary © by author

Invitation| Webversion

New Frontiers in Innate Immunity and Inflammation Cluj-Napoca, 5-7th September 2018 www.radboudumc.nl/newfrontiersininnateimmunityandinflammation

The ‘Iuliu Hatieganu’ University of Medicine and Pharmacy, the Romanian Center for Syst emsESCMID Immunology, and Radboud Center for Infectious D iseaeLibraryses Nijmegen invites you on Sept. 5th-7th 2018 to the international symposium ‘New Frontiers in Innate Immunity and Inflammation’ in the city of Cluj-Napoca, the heart of Transylvania. © by author