IL-7-dependent chemokine production by intestinal endothelial cells during acute HIV/SIV infection

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IL-7-dependent chemokine production by intestinal

endothelial cells during acute HIV/SIV infection

Carolina Moraes-Cabe, Magali Rancez, Bénédicte Charmeteau-De-Muylder,

Suzanne Figueiredo, Magali Mas, Rémi Cheynier, Anne Couëdel-Courteille

To cite this version:

Carolina Moraes-Cabe, Magali Rancez, Bénédicte Charmeteau-De-Muylder, Suzanne Figueiredo,

Ma-gali Mas, et al.. IL-7-dependent chemokine production by intestinal endothelial cells during acute

HIV/SIV infection. 20th International Vascular Biology Meeting, Jun 2018, Helsinki, Finland.

�hal-03000430�

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IL-7-dependent chemokine production by intestinal endothelial cells during acute

HIV/SIV infection

1_{INSERM, U1016, Institut Cochin, Paris 75014, France;}2_{CNRS, UMR8104, Paris 75014, France;}3_{Université Paris Descartes, Sorbonne Paris Cité, Paris 75014, France;}4_{Université Paris Diderot, Paris 75013, France}

Carolina Moraes-Cabe1,2,3_{, Magali Rancez}1,2,3_{, Bénédicte Charmeteau-de-Muylder}1,2,3 _{, Suzanne Figueiredo}1,2,3_{, Magali Mas}1,2,3_{, Rémi Cheynier}1,2,3_,

Anne Couëdel-Courteille1,2,3,4

Interleukin-7 (IL-7) is an essential cytokine for the development and homeostasis of T lymphocytes. This cytokine has long been described as constitutively produced by stromal cells of lymphoid and non-lymphoid organs. Homeostatic production of IL-7 is independent of extrinsic stimuli, plasma IL-7 levels being regulated by its consumption by T-cells (Fry and Mackall 2005, Mazzucchelli and Durum 2007). However, IL-7 production by stromal cells can be regulated by the microbiota (Shalapour et al., 2010).

Moreover, in vivo, we recently showed that IL-7 production is transiently increased in the gut of acutely SIV-infected rhesus macaques (Ponte et al., 2017), suggesting a regulation by inflammatory cytokines. This increase leads to local expression of several chemokines and immune cell homing into the gut, with a possible consequence on both the initiation of the antiviral immune response and the establishment of viral reservoirs (Ponte et al., 2017). Among all cells that compose the gut mucosa, endothelial cells play a key role in immune cell migration into tissues. These cells support transmigration, express chemokines upon stimulation (Nakano et al., 2012, Lo HM et al., 2014) and can produce IL-7 (Iolyeva et al., 2013). However, their role in IL-7 dependent T-cell homing during the acute phase of HIV/SIV infection remains to be addressed.

INTRODUCTION

ACKNOWLEDGMENT

This work was supported by ANRS, SIDACTION, INSERM and Université Paris Descartes. Thank you to IDMIT animal caretakers and veterinaries, to core facilities (Cybio and Genom’IC platforms at Cochin Institute) and other collaborators for additional support.

METHODOLOGY

AIMS

This work aims at deciphering the implication of endothelial cells (EC) in the IL-7-dependent chemokine expression observed in the gut of acutely SIV-infected macaques.

CONCLUSIONS

• CD127 expression is increased in EC upon stimulation by TNFa with higher expression when IL-7 is added in human EC derived from blood progenitors.

• In combination with TNFa, IL-7 triggers increase in mRNA expression of CCL4, CCL5 in macaque intestinal EC and CCL2, CCL4 and CCL5 in human EC, while only increasing CXCL8 proteins in intestinal macaque or CXCL10 proteins in human endothelial cells compared to TNFa stimulation alone after overnight culture. Among the chemokines overexpressed in the gut mucosa of both SIV infected and IL-7 macaques, only CCL4 and CXCL8 were expressed upon IL-7 stimulation of TNF-stimulated endothelial cells, suggesting that other cell types participate to the IL-7-dependent chemokine expression in vivo. Moreover, CXCL8 levels in the culture supernatants were lower in TNF-stimulated human endothelial cells derived from blood progenitors than in simian primary intestinal endothelial cells, suggesting a specific capacity of the latter to respond to IL-7 through IL-8 expression.

• During acute phase of SIV-infection, as a result of the inflammatory environment and the IL-7 production, endothelial cells are likely to participate to the increase of chemokine production that leads to immune cell recruitment.

RESULTS

M055

Endothelial cells differentiated from blood progenitors Endothelial cells sampled from enzymatic digested macaque gut

mucosa Inflammatory Stimulation CCL2, CCL3, CCL4, CCL5, CCL19, CCL20, CCL25, CCL28, CXCL8, CXCL10, CXCL12, CX3CL1. Chemokine production analysis by RT-qPCR and ELISA

IL-7Ra (CD127) is expressed on endothelial cells and up-regulated upon stimulation

Fig 1. IL-7Ra (CD127) is overexpressed in stimulated endothelial cells

CD127 mRNAs expression in human (A) and macaque (B) EC was quantified by RT-qPCR in non-stimulated and 4H(A) or overnight stimulated (B) cells.

Fig 6. Correlation between chemokine concentrations and chemokine mRNA levels in stimulated human endothelial cells.

CCL2, CCL4, CCL5 and CXCL10 mRNAs were quantified by RT-qPCR and normalized to HPRT mRNA and results are express as chemokine mRNA copies/HPRT mRNA copy. CCL2, CCL4, CCL5 and CXCL10 concentrations were assessed by ELISA (MSD) and results are express as pg/mL. Regression line, Spearman’s rank correlation value, and associated probability are shown. 1000 10000 100000 1000000 0, 1 1 10 100

CCL2 mRNA copies/HPRT mRNA copy

A – CCL2 1 10 100 1000 10000 0, 001 0, 01 0, 1 1 10

B – CCL5 p=0.0369 Rho=0.629 p=0.0204 Rho=0.699 CCL 2 (pg /mL ) CCL 5 (pg /mL ) 1 10 100 1000 0, 0001 0, 001 0, 01 0, 1

C – CCL4 10 100 1000 10000 100000 0, 001 0, 01 0, 1 1 10

CXCL10 mRNA copies/HPRT mRNA copy

D – CXCL10 p=0.0514 Rho=0.587 p=0.0217 Rho=0.692 CCL 4 (pg /mL ) CX CL 10 ( pg /mL )

mRNA expression correlate together with protein production of CCL2, CCL4,

CCL5 and CXCL10 in human

endothelial cells IL-7 potentiates the effect of inflammatory cytokines for the expression

of some chemokines in human endothelial cells

Fig 4. Chemokines transcription is up-regulated in stimulated endothelial cells

mRNAs coding for CCL2 (A), CCL4 (B), CCL5 (C), CCL20 (D), CXCL8 (E) and CXCL10 (F) in non-stimulated and 4Hstimulated human EC were quantified by RT-qPCR.

Fig 5. Chemokine production in the culture supernatants of stimulated human endothelial cells.

CCL2 (A), CCL4 (B), CCL5 (C), CCL20 (D), CXCL8 (E) and CXCL10 (F) concentrations in non-stimulated and overnight stimulated human EC culture supernatants were assessed by ELISA (MSD).

0 2 4 6 8 10 12 14 16 18 20 CCL 2 ex pr es sio n (fo ld ch an ge fr om N S) 0 50 100 150 200 250 300 350 CCL 5 ex pr es sio n (fo ld ch an ge fr om NS ) 0 5 10 15 20 25 30 35 40 CCL 4 ex pr es sio n (fo ld ch an ge fr om NS ) A – CCL2 B – CCL4 C – CCL5

NS IL-7 TNF TNF+IL-7 NS IL-7 TNF TNF+IL-7 NS IL-7 TNF TNF+IL-7

0.02 0.02 0.02 0.02 0.02 0.02 0.04 0.02 0.02 0.02 0.02 0.02 0.04 0.02 0.02 150 160 170 180 0 10 20 30 40 50 0 5 10 15 20 25 CX CL 8 ex pr es sio n (fo ld ch an ge fr om N S) 0 30 60 90 120 150 CCL 20 e xp re ss io n (fo ld ch an ge fr om NS ) D – CCL20 E – CXCL8 F – CXCL10

CX CL 10 e xpr es sio n (fo ld ch an ge fr om N S) 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.05 0.05 0.05 0.05

IL-7 potentiates the effect of inflammatory cytokines for the expression of some chemokines in macaque endothelial cells

A – CXCL8 0,1 1,0 10 ,0 10 0,0 1 00 0,0 NS IL-7 TNF TNF+IL-7 0,1 1,0 10 ,0 10 0,0 CX CL 8 (F ol d Ic ha ng e fro m N S) CX CL 10 (F ol d Ic ha ng e fro m N S) NS IL-7 TNF TNF+IL-7 B – CXCL10 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Fig 3. Chemokine concentrations in the culture supernatants of stimulated simian endothelial cells.

CXCL8 (A) and CXCL10 (B) concentrations in non-stimulated and overnight stimulated simian EC culture supernatants were assessed by ELISA (MSD).

Each symbol represents one donor. Results are presented as fold increases over non-stimulated (NS) cells values or cell culture supernatant values. RT-qPCR are normalized to HPRT mRNA. Statistical analysis were made using Wilcoxon Signed-Rank Test for Paired Samples.

0, 1 1, 0 10, 0 100, 0 CD 12 7 ex pr es sio n (fo ld ch an ge fr om N S) A - CD127 (human EC) NS IL-7 TNF TNF+IL-7 0.04 0.04 0.04 0.04 0, 1 1, 0 10, 0 100, 0 CD 12 7 ex pr es sio n (fo ld ch an ge fr om N S) NS IL-7 TNF TNF+IL-7 B - CD127 (macaque EC) 0.07 0.07 0.07 0.07 0.04

Fig 2. Chemokines transcription is up-regulated in stimulated simian endothelial cells

mRNAs coding for CCL4 (A), CCL5 (B) and CXCL10 (C) in non-stimulated and overnight stimulated simian EC were quantified by RT-qPCR. 0.05 22 25 28 0 4 8 12 16 NS IL-7 TNF TNF+IL-7 CX CL 10 (F ol d cha ng e fro m N S) C – CXCL10 CX CL 10 (F ol d cha ng e fro m N S) 0 2 4 6 8 10 12 14 16 A – CCL4 NS IL-7 TNF TNF+IL-7 CCL 4 (F ol d cha ng e fro m N S) 0.05 0.05 0.05 0.05 0 40 80 120 4 500 5 000 5 500B – CCL5 CCL 5 (F ol d cha ng e fro m N S) NS IL-7 TNF TNF+IL-7 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 B – CCL4 A – CCL2 CCL 5 (F ol d cha ng e fro m N S) 0 50 100 150 200 250 600 700 800 900 0.05 0.05 0.05 0.05 0 5 10 15 20 25 30 35 CCL 4 (Fo ld ch an ge fr om NS ) 0 5 10 15 20 25 30 35 40 CCL 2 (Fo ld ch an ge fr om NS ) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

D – CCL20 E – CXCL8 F – CXCL10 0 5 10 15 20 25 CX CL 8 (Fo ld ch an ge fr om NS ) 0.05 0.05 0.05 0.05 CCL 20 (F ol d cha ng e fro m N S) 0 5 10 15 20 25 30 35 0.05 0.05 0.05 0.05

NS IL-7 TNF TNF+IL-7 NS IL-7 TNF TNF+IL-7 0

30 60 90 120 600 650 700 0.05 0.05 0.05 0.05 0.05 CX CL 10 (F ol d cha ng e fro m N S) NS IL-7 TNF TNF+IL-7 C – CCL5

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