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GUT MUCOSAL IL22+ T CELLS ARE RELATED WITH INCOMPLETE CD4 RESTORATION DESPITE cART

Financiación: Fondo de Investigacion Sanitaria, Instituto de SaludCarlos III [PI14/01693 and PI18/1216] and Consejería de Economía,Innovación, Ciencia y Empleo, Junta de Andalucía (Proyecto deInvestigación de Excelencia; CTS2593). YMP is supported by grantfrom the Servicio Andaluz de Salud (Nicolás Monardes C-0013-2017)

Rosado-Sánchez I1, Herrero-Fernández I1, Sobrino S2, Carvajal AE1,3, Genebat M1, de Pablos RM1,3, Ruiz R1,3, Sánchez-Villegas J4, Leal M1,5 and Pacheco YM1

Gut mucosal immunity plays a central role in the HIV pathogenesis but still large gaps of knowledge remain. In the scenario of the incomplete CD4-recovery, impaired gut junctionalcomplexes and increased markers of intestinal permeability and damage have been described. However, a functional assessment of T-cells has not been yet performed in such scenario,despite the well-known role of cytokines like IL22 in mucosal integrity, and the fact that CD4 T-cells may also contribute to the production of this cytokine.

AIM - To explore gut mucosal CD4 T-cell function and its potential relationship with the mucosal damage and immune reconstitution.

Background

1Institute of Biomedicine of Seville, IBiS, Virgen del Rocío University Hospital/CSIC/University of Seville, Seville, Spain; 2Digestive Service, Virgen del Rocío University Hospital; 3Department of Biochemistry and Molecular Biology, University ofSeville, Seville, Spain; 4Hospital Comarcal de la Merced, Osuna, Sevilla, Spain; 5Internal Medicine Service, Viamed-Santa Ángela Hospital, Seville, Spain.

Parameters

for T. Ileum

Intestinal

villi

Goblet cells Crypt

dilatation

Paneth

cells

Mucosal

infiltration

SCO

RE

0 Glove

finger

Normal

appearance

Normal

morphology

Normal

appearance

Normal

1 Slightly

widened

and

shortened

Slightly

reduced

Slightly

dilated /

local zone

Slightly

reduced

Slightly

increased

2 Moderately

widened

and

shortened

Moderately

reduced

Moderate

dilatation /

several

zones

Moderately

reduced

Moderately

increased

3 Severely

widened

and

shortened

Severely

reduced or

absent

Severe

dilatation /

most zones

Severely

reduced or

absent

Severely

increased

Methods

Biopsies of both, caecum (C) and terminal ileum (TI) of non-HIV healthy subjects (n=6)and treated, virally-suppressed, HIV-infected subjects were obtained: subjects withCD4 T-cell counts bellow 250 cell/ul after two years of suppressive-treatment (INR,n=9) and control subjects overcoming such threshold (IR, n=7). Histological assessmentof mucosal damage was performed using a semi-quantitative scale of five physicalparameters (*). MMCs were digested, isolated and stimulated (with PMA/Iono) toquantify the T-cell production of different cytokines by flow-cytometry. Th22, Th17,Th1 and Treg cells were analyzed. In a subset of subjects, the expression of mucosalcaspase-3, gal-3, Zo-1 and mucin was also analyzed by immunofluorescence. LBPlevels, as a surrogate marker for intestinal permeability, was measured by ELISA inperipheral samples. Comparisons among groups were explored by the Kruskall-Wallisand Mann-Withney U tests, and correlations by the Spearman rank test. Collagenase

Digestion

MMCsIsolation

Parameters

for Caecum

Epithelial

destruction

Crypt/Gland

destruction

Crypt

dilatation

Goblet loss

or

reduction

Mucosal

infiltration

SCO

RE

0 Normal

morphology

Normal

morphology

Normal

morphology

Normal

appearance

Normal

1 Local

destruction

Local

destruction

Slightly

dilated /

local zone

Slightly

reduced

Slightly

increased

2 Zonal

destruction

Zonal

destruction

Moderate

dilatation /

several

zones

Moderately

reduced

Moderately

increased

3 Extensive

destruction

Extensive

destruction

Severe

dilatation /

most zones

Severely

reduced or

absent

Severely

increased

Terminal ileum

Caecum

ypacheco-ibis@us.es

(*) HISTOLOGICAL ASSESMENT

IMMUNOFLUORESCENCE

FLOW CYTOMETRY(MMCs)

SOLUBLELBP

GALT BIOPSIES

PMA/Ionostimulation

Results

Subjects with incomplete CD4-recovery show reduced capacity of gut mucosal T-cells to produce IL22. This cytokine, with a dual “inflammatory-protective” role during tissue responsesto inflammation, could have a protective-regenerative potential on the gut potentially necessary for the normal CD4-recovery.

Conclusion

0) Characteristics of studied subjects1) INR showed the highest mucosal damage atboth locations

2) The frequencies ofIL22+CD4+ mucosal T-cells correlated withperipheral CD4 T-cellcounts (rho=0.738,p<0,001 for caecum;rho=0.682, p=0.001 forterminal ileum)

3) INR showed the lowest frequencies of IL22+CD4+mucosal T-cells, whereas the highest IL17+/IL22+CD4mucosal T-cells ratios, independent of the location

4) INR showed the highest frequencies of mucosalTreg (particularly at TI), whereas the lowestIL22+CD4+/Treg mucosal T-cells ratios, independent ofthe location

5) IL22+CD4+/Treg mucosal T-cell ratios inversely correlated with mucosal damage(rho= -0.579, p=0.015 at caecum; rho=-0.863, p<0.0001 at terminal ileum)

6) At terminal ileum, the frequency of IL22+CD4+mucosal T-cells correlated with in situ markers of mucosalintegrity and with a peripheral surrogate marker ofbacterial translocation (LBP)

Healthy (n=6) IR (n=7) INR (n=9)

Age (years) 54 [46-63] 48 [45-66] 54 [51-56]

Male sex, n/n (%) 5/6 (83) 7/7 (100) 6/9 (67)

CD4 (cells/mm3) 832 [734-858] 560 [418-756] 251 [180-312]

CD4/CD8 T-cell ratio 2.83 [1.55-4.29] 0.95 [0.55-1.68] 0.42 [0.24-0.58]

Nadir CD4 (cells/mm3) N.A. 39 [13-137] 18 [3-39]

Zenith viral load (log HIV RNA copies/mL)

N.A. 5.11 [5.00-5.70] 5.23 [4.91-5.38]

Time under virologicalsuppression (years)

N.A. 8 [6-13] 6 [5-13]

FOOTNOTES: Values are given as median [IQR] unless otherwise indicated. N.A. Not Applicable

% IL 2 2 + C D 4 + (C )

Healt

hy IR

INR

0

2 0

4 0

6 0

8 0

K -W p = 0 .0 0 7

p = n s p = 0 .0 9 1

IL 1 7 a + / IL 2 2 + C D 4 + (C )

Healt

hy IR

INR

0

5

1 0

1 5

2 0

K -W p = 0 .0 1 1

p = n s p = 0 .0 4 2

% IL 2 2 + C D 4 + (T I)

Healt

hy IR

INR

0

2 0

4 0

6 0

8 0

K -W p = 0 .0 0 3

p = n s p = 0 .0 1 6

IL 1 7 a + / IL 2 2 + C D 4 + (T I)

Healt

hy IR

INR

0

5

1 0

1 5

2 0

K -W p = 0 .0 1 6

p = n s p = 0 .0 5 4

T r e g M iy a r a (C )

Healt

hy IR

INR

0

5

1 0

1 5

2 0

K -W p = n s

IL 2 2 + C D 4 + / T r e g (C )

Healt

hy IR

INR

0

1 0

2 0

3 0

4 0

1 5 0

2 0 0

2 5 0

K -W p = 0 .0 0 5

p = n s p = 0 .0 0 6

T r e g M iy a r a (T I )

Healt

hy IR

INR

0

5

1 0

1 5

2 0

K -W p = 0 .0 0 5

p = n s p = 0 .0 0 8

IL 2 2 + C D 4 + / T re g (T I)

Healt

hy IR

INR

0

2 0

4 0

6 0

1 0 0

1 5 0

2 0 0

2 5 0

K -W p = 0 .0 0 1

p = 0 .0 7 3 p = 0 .0 0 1

c lv -C a s p -3 (% o f H o e c h s t )

% I

L2

2+

CD

4+

0 2 0 4 0 6 0

0

2 0

4 0

6 0

rh o = -0 .5 9 3

p = 0 .0 3 3

Z O -1 (% o f H o e c h s t )

% I

L2

2+

CD

4+

0 5 0 1 0 0 1 5 0

0

2 0

4 0

6 0

rh o = -0 .6 2 6

p = 0 .0 2 2

G a l-3 (% o f H o e c h s t )

% I

L2

2+

CD

4+

0 2 0 4 0 6 0 8 0

0

2 0

4 0

6 0

rh o = 0 .6 4 6

p = 0 .0 0 9

L B P

% I

L2

2+

CD

4+

5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0

0

2 0

4 0

6 0

8 0

rh o = -0 .4 5 4

p = 0 .0 5 1