Poster number INSERM U1043 Team 6 Toulouse University Hospital 0203 Synergy between Th1 and Th22 impairs Th17 cells recruitment to the gut on ART 31059 Toulouse, FRANCE E-mail: [email protected] Manon Nayrac1, Mary Requena2, Claire Loiseau3, Maud Mavigner4, Fatima-Ezzahra L’Faqihi-Olive1, Nicolas Carrere2, Bertrand Suc2, Jacques Izopet1,2, Pierre Delobel1,2 Phone: +33 5 61 67 69 04 57 1U1043, INSERM, Toulouse, France, 2 Toulouse University Hospital, Toulouse, France, 3 Australian Institute of Tropical Health and Medecine, Cairns, Australia, 4 Emory Vaccine Center, Atlanta, GA, USA

INTRODUCTION : During HIV-1 infection, a deep depletion of Th17 cells occurs early in the gut mucosa. Th22 cells are also CCL20-CCR6 and CCL28-CCR10 chemotactic axes in the duodenal mucosa Cooperation between Th1 and Th22 cells in intestine mucosa initially depleted but appear to be able to efficiently recover on antiretroviral therapy (ART), while Th17 do not. In-vivo CCL20 CCL28 A pro-inflammatory state also promotes Th1 cells recruitment to the gut on ART. Both Th17 and Th22 cells express CCR6 and could thus be recruited through the CCL20-CCR6 axis. However, we previously HIV-1 - reported that CCL20 production by enterocytes is impaired in the duodenum on ART. But Th22 cells can alternatively use the

x20 18 mRNA18 CCL28-CCR10 axis to migrate to the gut. x200 x200 - IL We hypothesized that Th1 and Th22 cells synergistically impair CCL20 production by the enterocytes thus preventing Th17 cells change expression (A.U) changeexpression - CCL20 mRNA mRNA CCL20 recruitment to the gut. -18

CCL28 expression expression CCL28 18 fold

change expression (A.U) changeexpression - CCL20 expression expression CCL20 per surface unit (%) unit surface per 18 -

per surface unit (%) unit surface per - HIV-1 + γ - γ -γ 50 ng/mL IL - fold HIV-1 - + HIV-1 - + -22 -22 -22 5 ng/mL IL 0.5 ng/mL IL METHODS : Duodenal biopsies were obtained from 10 HIV-1-infected subjects on ART and 10 uninfected controls. Intestinal T 5 ng/mL IFN 5 ng/mL IL x200 x200 0.5 ng/mL IFN 50 ng/mL IFN0.5 ng/mL IL 50 ng/mL IL cells were isolated and Th1 (CD3+CD4+CXCR3+CCR4-CCR6-), Th17 (CD3+CD4+CXCR3-CCR4+CCR6+CD161+), and Th22 Figure 5. IL-18 expression by enterocytes after ex-vivo Figure 6. Impact of IL-18 on CCL20 expression by + + - + + - + Figure 2. Expression of CCL20 and CCL28 by enterocytes in the duodenum of HIV-1-infected subjects vs. uninfected controls (CD3 CD4 CXCR3 CCR4 CCR6 CD161 CCR10 ) cell frequencies were analyzed by flow cytometry (BD Fortessa). stimulation by IFN-γ or IL-22, relatively to unstimulated enterocytes, relatively to unstimulated A model of primary human intestine epithelial cell (IEC) culture was used to decipher enterocyte response to and T cells In contrast to the reduced production of CCL20 by enterocytes in HIV-1-infected subjects, that blunts Th17 gut homing, CCL28 IFN-γ and IL-22 increased IL-18 expression (respectively up to 1000- and 10-fold), which in turn further decreased CCL20 in co-culture experiments. CCL20 and CCL28 expression was quantified by qRT-PCR (mRNA) and ELISA (protein). production was maintained in the duodenal mucosa allowing Th22 recruitment through the CCR10 axis in this setting. expression by 2 to 3-fold.

RESULTS: CCL20 and CCL28 expression by enterocytes Ex-vivo

* * FSC CCR4 change expression changeexpression toTh17:IEC (A.U) fold mRNA

relatively * CCL20 CCL20

CD4 mRNA CCL20 18BP CXCR3 - -22BP

CCL28 CCL28 18BP - -22BP change expression (A.U) changeexpression -

change expression (A.U) changeexpression Th22:IEC - Th1:IEC fold Th22:IEC + IL

fold Th22:IEC + IL γ γ 2 α 21 α 17F Th1:IEC +Th1:IEC IL + IL - 17A -17F - -22 -2 17A - -21 -22 + - - IFN - IFN - IL Th22: CXCR3-CCR4+CCR6+CCR10+ Th17: CXCR3-CCR4+CCR6+CD161+ Th1: CXCR3 CCR4 CCR6 IL TNF IL IL IL IL IL TNF IL IL IL Figure 7. Impact on CCL20 expression of antagonizing IL-18 and IL-22 biological effect in Th1:IEC and Th22:IEC cocultures, relatively to Th17:IEC (P<0.05) Th1 Th17 Th22 Th1 Th17 Th22 cytokines cytokines cytokines cytokines cytokines cytokines The negative impact of Th22 and Th1 cells on CCL20 expression by enterocytes involves IL-18 and IL-22. CD4 CCR6 CCR6 Figure 3. Expression of CCL20 and CCL28 mRNA by IEC after ex-vivo stimulation by Th cytokines (5 ng/mL) Fold-change expression relatively to unstimulated CONCLUSION : Th1 and Th22 synergistically blunt CCL20 expression by enterocytes through IFN-γ, IL-18, and IL-22, Ex-vivo, IFN-γ and IL-22, respectively the main Th1 and Th22 , induced a 5-fold and 2.5-fold decrease in CCL20 mRNA preventing Th17 reconstitution in the gut of HIV-1-infected subjects on ART. CCR6 CCR10 CD161 expression by enterocytes. By contrast, CCL28 expression was not significantly modulated by any Th cytokines.

P<0.01 P=0.02 P=0.53 P<0.05 P<0.01

active IL-1β active IL-1β active IL-18 active IL-18 proIL-18 proIL-18 il-18 proIL-1β il-18 proIL-1β ccl28 ccl20 ccl28 Gut (%) Th1 cells Gut (%) Th17 cells Gut (%) Th22 cells il-1β il-1β change expression changeexpression -

HIV-1 - + HIV-1 - + HIV-1 - + IL-17 CCL20 Th1 IL-22 relativelytoTh17:IEC (A.U) CCR6 CCL28 CCL28 Figure 1. Gating strategy for gut T cell subsets analysis relativelytoTh17:IEC (A.U) Th1:IEC Th22:IEC IFNγ CCL20 fold CCL20 Th17 Th17 CCL20 CCR10 IL-17 CCR10 CCL28 fold change expression expression change fold CCL28 Th1 Th1:IEC Th22:IEC Th22 Th22 Th22 IFNγ The frequency of Th22 cells in the duodenum of treated HIV-1-infected subjects was restored to normal values (6.3% vs 5.4%, CCR6 Th22 IL-22 Th17 Th1 Th1 Th1 P=0.53). By contrast, Th17 cells remained lower than in uninfected controls (4.3% vs 7.6%, P<0.05), and Th1 cells were increased Th17 Figure 4. Expression of CCL20 and CCL28 in Th1:IEC and Th22:IEC cocultures, relatively to Th17:IEC Th22 Th17 Th17 Th1 Th22 (9.0% vs 4.7%, P<0.01) in HIV-1-infected subjects vs uninfected controls. Th17 IL-22 Th1 and Th22 cells both induced a lower expression of CCL20 by enterocytes than Th17 cells. By contrast, Th1 and Th22 cells did Th1 Th1 Th17 not change CCL28 expression by enterocytes compared to Th17 cells, in agreement with cytokine results. Homeostatic Inflammatory environment environment