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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
(*) 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