The Journal of Infectious Diseases Phenotype and polyfunctional deregulation involving IL-6- and IL-10-producing monocytes of HIV-infected patients on cART differ from those of aging --Manuscript Draft-Manuscript Number:
58165R2
Full Title:
Phenotype and polyfunctional deregulation involving IL-6- and IL-10-producing monocytes of HIV-infected patients on cART differ from those of aging
Short Title:
Monocyte ageing and chronic HIV
Article Type:
Major Article
Section/Category:
HIV/AIDS
Keywords:
HIV; inflammation; monocyte; aging
Corresponding Author:
Ezequiel Ruiz-Mateos Institute of Biomedicine of Seville (IBIS) Seville, Sevilla SPAIN
Corresponding Author Secondary Information: Corresponding Author's Institution:
Institute of Biomedicine of Seville (IBIS)
Corresponding Author's Secondary Institution: First Author:
RS De Pablo-Bernal
First Author Secondary Information: Order of Authors:
RS De Pablo-Bernal R Ramos M Genebat J Cañizares M R Benhnia MA Muñoz-Fernández Y M Pacheco M I Galvá M Leal Ezequiel Ruiz-Mateos
Order of Authors Secondary Information: Manuscript Region of Origin:
SPAIN
Abstract:
Background: Despite the relevance of monocytes as promoters of the inflammatory response, whether HIV infection induces premature age-related changes to the phenotype and function of monocytes or these alterations are different and/or specifically driven by HIV remains to be mechanistically determined. Methods: We assayed the activation phenotype and the responsiveness in vitro to TLR agonists in classical, intermediate and non-classical subsets of monocytes by assessing the intracellular IL-1α, IL-1β, IL-6, IL-8, TNF-α and IL-10 production in patients on cART (n=20, HIV) compared to two groups of uninfected controls (agematched, n=20, Young or over 65 years old, n=20, Elderly). Results: HIV-infected patients showed a more activated phenotype of monocytes than the elderly. Regarding functionality, under unstimulated conditions, the HIV group showed a higher percentage of classical monocytes producing IL-6 and IL-10 compared with controls. The percentage of cells with multiple cytokine production
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(polyfunctionality) in response to TLR agonists in the HIV group, specially with IL-10, was higher than in the controls. Conclusion: Inflammatory alterations associated with monocytes in HIV infection are different from those of aging. This monocyte dysfunction, mainly characterized by high levels of IL-6- and IL-10-producing monocytes, may have clinical implications in HIVinfected patients different from those of aging.
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Cover Letter
Seville, 6th October, 2015
Professor Martin S. Hirsch, Please find enclosed the revised version of the manuscript entitled “Phenotype and polyfunctional deregulation involving IL-6- and IL-10-producing monocytes of HIV-infected patients on cART differ from those of aging”. The authors want to thank the editor for the recommendations. We agree with the editorial comments and we really think these recommendations will improve the quality of the study. Therefore, we have endeavored in clarifying the seven points in the new revised version of the manuscript. We hope that the work is now suitable for publication in The Journal of Infectious Diseases.
All of the authors have contributed to, read and approved the final version of the manuscript.
Sincerely,
Ezequiel Ruiz-Mateos PhD, Corresponding author. Immunovirology Laboratory (Lab 211), Biomedicine Institute of Seville (IBIS). Virgen del Rocío University Hospital. Avda. Manuel Siurot s/n. CP 41013, Seville, Spain.
[email protected]
Instituto de Biomedicina de Sevilla – IBiS Campus Hospital Universitario Virgen del Rocío Avda. Manuel Siurot, s/n - 41013 Sevilla Tel. 955923000 - Fax 955923101 www.ibis-sevilla.es
Response to Editor/Reviewer Comments Click here to download Response to Editor/Reviewer Comments: 20151006_comments.docx
Professor Martin S. Hirsch, In agreement with your suggestions for the manuscript entitled “Phenotype and polyfunctional deregulation involving IL-6- and IL-10-producing monocytes of HIV-infected patients on cART differ from those of aging” we had addressed the seven points of the decision letter waiting our answers could further clarify those pitfalls in order to make the manuscript suitable for publication in the Journal of Infectious Diseases.
Comments to the Author Editorial comments: Your revised manuscript is improved. However, there are additional comments, listed below, that require your consideration in the further revision of your manuscript We really want to thank the editor’s consideration and recommendations that will allow a better interpretation of the results by the readers. 1. On lines 265-8 and lines 272-6, as well as supplemental fig. 3, please clarify if the associations are for all the patients (both HIV-infected and uninfected) or for subgroups. If for the group as a whole, were similar associations found for the subgroups? In relation to the associations described in lines 265-8 and supplementary figure 3, we have explained in the text and in the figure legend that the associations are for the whole group of subjects (n=60). In addition, we have included a detailed explanation of the results when splitting the participants into the three groups of study: “However, most of these associations were lost after stratification into groups (young, HIV and elderly), probably due to the small number of studied cases. Interestingly, the analysis of the HIV group separately revealed strong associations only significant for this subgroup. There was a strong inverse correlation between sCD14 levels and the IL-10 production (p=0.009, rho= -0.594) in the intermediate subset without stimulation and between the sCD163 levels and the IL-8 production in intermediate and non-classical subsets of monocytes also, in the unstimulated condition (p=0.003, rho= -0.629; p=0.016, rho= -0.531, respectively) of HIV-infected patients” Regarding correlations in lines 272-6, we have clarified that the associations shown are for the three separately groups. In addition, we have pointed out, that the most significant and strongest associations between these markers were found for the HIV group.
2. In the legend to figure 3, please indicate that the values shown represent the difference between the stimulated and unstimulated values. The authors regret if the caption of figure 3 was not adequately explained. We have added the required information in the figure 3 legend of the new version of the manuscript. 3. The phrase "ex vivo" (line 145 an elsewhere) does not seems to be appropriate, since all the cells are stimulated with anti-CD28 and anti-CD49b for 6 hours and, thus, are being activated while being cultured for 6 hours. Probably the term “ex vivo” does not resemble properly the methodology. Therefore, in agreement with the editor’s recommendation, we have replaced the ex vivo term to the more appropriate term: “unstimulated” in line 145 and elsewhere in the new version of the manuscript. 4. In supplemental fig. 1, the phrase in the figure legend that the non-classical monocyte subset was gated correctly despite the shedding of CD16 remains confusing since it is clear that CD16 is still being expressed and gated upon. It might be simpler to delete this phrase or, alternatively, to note that conditions were utilized that minimized shedding of CD16 and allowed identification of that subset. We agree the sentence lead to a confusing interpretation. Thus we have replaced our phrase for the editor’s second suggestion: “Note that the conditions utilized minimize the shedding of CD16 and allowed identification of the non-classical subset” in the text (lines 227-8) and in the figure legend of supplementary figure 1.
5. Table 2 needs to be a figure, rather than a table, if some cells are colored in. We regret for this form defect and change the colored cells for bold values to highlight significant differences in all the tables of the new version of the manuscript.
6. For Supplemental table 1, does the MFI represent the positive population only, or is it the MFI for the entire population of cells? The MFI values represent only the positive population. Since at least the 0.01% of each monocyte subset produced cytokines among all groups (young, HIV and elderly), there is a positive MFI value (always considering a minimum of gated events which make a feasible measurement). This point has been added to the manuscript.
7. For Supplemental table 3, are the differences (values above the unstimulated) calculated using the arithmetic or geometric values? MFI is usually expressed on a log scale, so it would be more appropriate to use log values.
Data are expressed as median not as geometric or arithmetic mean values, we use median values since this is a more robust and less affected by outliers measurement. The expression of the median intensity fluorescence is expressed in absolute numbers, because there are many values that are 0 and the log value of 0 is not defined. For this reason, we consider the expression of the values as absolute numbers the most accurate option in this case.
Manuscript Click here to download Manuscript: 20151006_DePabloetalmanuscript_R2.docx
1
Title: Phenotype and polyfunctional deregulation involving IL-6- and IL-10-
2
producing monocytes of HIV-infected patients on cART differ from those of aging
3 4
De Pablo-Bernal RS1, Ramos R2, Genebat M1, Cañizares J2, Benhnia MR1,3, Muñoz-
5
Fernández MA4, Pacheco YM1, Galvá MI2, Leal M1#, Ruiz-Mateos E1#*
6 7 8 9 10 11
#
these authors contributed equally to this work
1
Laboratory of Immunovirology, Clinic Unit of Infectious Diseases, Microbiology and
12
Preventive Medicine, Institute of Biomedicine of Seville, IBiS, Virgen del Rocío University
13
Hospital/CSIC/University of Seville, Seville, Spain
14
2
Heliopolis nursing home, Seville, Spain
15
3
Department of Biochemistry and Molecular Biology & Immunology, Medical School,
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University of Seville, Seville, Spain
17
4
18
Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
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Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-
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BBN), Madrid, Spain.
Laboratory of Molecular Immuno-Biology Hospital General Universitario Gregorio
21 22 23
Running head: Monocyte aging and chronic HIV
24
Text word count: 3418
25
Abstract word count: 196
26 27 28 29
*Correspondence: Ezequiel Ruiz-Mateos, (PhD), Immunovirology Laboratory (Lab 211), Institute of Biomedicine of Seville, Virgen del Rocío University Hospital, Avda Manuel Siurot s/n CP 41013, Seville, Spain (
[email protected]) +34955923107.
30
ABSTRACT:
31
Background: Despite the relevance of monocytes as promoters of the inflammatory response,
32
whether HIV infection induces premature age-related changes to the phenotype and function of
33
monocytes or these alterations are different and/or specifically driven by HIV remains to be
34
mechanistically determined.
35
Methods: We assayed the activation phenotype and the responsiveness in vitro to TLR agonists
36
in classical, intermediate and non-classical subsets of monocytes by assessing the intracellular
37
IL-1α, IL-1β, IL-6, IL-8, TNF-α and IL-10 production in patients on cART (n=20, HIV)
38
compared to two groups of uninfected controls (age-matched, n=20, Young or over 65 years
39
old, n=20, Elderly).
40
Results: HIV-infected patients showed a more activated phenotype of monocytes than the
41
elderly. Regarding functionality, under unstimulated conditions, the HIV group showed a higher
42
percentage of classical monocytes producing IL-6 and IL-10 compared with controls. The
43
percentage of cells with multiple cytokine production (polyfunctionality) in response to TLR
44
agonists in the HIV group, specially with IL-10, was higher than in the controls.
45
Conclusion: Inflammatory alterations associated with monocytes in HIV infection are different
46
from those of aging. This monocyte dysfunction, mainly characterized by high levels of IL-6-
47
and IL-10-producing monocytes, may have clinical implications in HIV-infected patients
48
different from those of aging.
49 50 51 52 53 54 55 56 57
Key words: HIV, inflammation, monocyte, aging
2
58
INTRODUCTION
59
Aging and HIV infection are associated with profound changes in the immune
60
system, inducing several similar defects associated with T-cell function [1, 2].
61
Additionally, higher levels of inflammatory and coagulation biomarkers have been
62
reported for HIV-infected patients on combined antiretroviral therapy (cART) and for
63
elderly uninfected subjects [3-5], which likely increase their risks for developing
64
chronic diseases [6, 7]. However, we have recently shown marked differences in the
65
inflammatory biomarker profile of cART-treated and HIV-uninfected elderly subjects
66
[8].Together, these findings beg the question of how the inflammatory mechanisms of
67
HIV infection and age intersect. In this sense, the latest studies have focused on better
68
understanding the changes in innate immune cells, revealing profound deregulations in
69
the blood mononuclear phagocyte system with aging and HIV infection [9, 10].
70
Human monocytes are innate immune cells characterized by a high degree of
71
heterogeneity and complexity. Monocytes comprise three subsets, the classical
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monocytes (CD14++CD16−), the intermediate monocytes (CD14++CD16+), and the
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non-classical monocytes (CD14dimCD16++), that differ in size, morphology,
74
phenotype and function. The classical and intermediate subsets secrete high levels of
75
pro-inflammatory cytokines in response to microbial products, and the non-classical
76
subset appears to patrol the vessel wall [11]. However, despite the fact that monocytes
77
are potentially the major source of the circulating inflammatory cytokines found in
78
HIV-infected patients and normal aging [12], mechanistically, the information about the
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single and multiple cytokine production (polyfunctionality) of these cells in both
80
scenarios has not been explored.
81
The aim of this study was to comprehensively analyze phenotypic and functional
82
characteristics of the three subsets of monocytes in cART-treated HIV infected patients
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and to compare them to those from the aging scenario.
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PATIENTS AND METHODS
85
Study participants
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Twenty Caucasian, asymptomatic, HIV-infected patients on suppressive cART
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(defined as persistent undetectable viral load for at least 6 months) that consecutively
88
visited the outpatient clinic of the Infectious Disease Unit at Virgen del Rocío
89
University Hospital in Seville (Spain) between May and July of 2014 were included.
90
Patients were excluded if they had a current or previous history of cardiovascular
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disease, diabetes mellitus, chronic kidney disease or had reached clinical category C of
92
HIV infection.
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HIV-infected patients (the HIV group, n=20) were compared with age-matched,
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HIV-uninfected healthy subjects, called the Young group (Young, n=20), and with
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elderly subjects, called the Elderly group (Elderly, n=20), who had already been studied
96
with a different objective [12] and served as the control groups for the present study.
97
Briefly, consecutive nursing home residents from Seville (Spain) were asked between
98
May and July of 2014 to participate in this cohort. The inclusion criteria were being
99
aged 65 years or older and having a self-sufficient health status. The exclusion criteria
100
included any of the following situations during the preceding six months: (1) clinical
101
data indicating active infections, (2) hospital admission, (3) anti-tumor therapy, or (4)
102
any treatment that could influence the immune status (mainly corticosteroids).
103
Laboratory evaluations were performed at the Laboratory of Immunovirology, Institute
104
of Biomedicine (IBiS), Virgen del Rocío University Hospital in Seville (Spain). All
105
necessary institutional or ethical review board approvals were obtained, and written
106
informed consent was obtained from all study participants.
107 108
Laboratory methods
109
The absolute CD4+, CD8+ T-cell counts (cells/mm3) and the CD4:CD8 ratio
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were determined using an Epics XL-MCL flow cytometer (Beckman-Coulter, Brea, CA,
111
USA) according to the manufacturer’s instructions. The plasma HIV-1 RNA
112
concentration (HIV-RNA copies/mL) was measured using quantitative polymerase
113
chain reaction (COBAS Ampliprep/COBAS Taqman HIV-1 Test, Roche Molecular
114
Systems, Basel, Switzerland) according to the manufacturer’s protocol. The detection
115
limit for this assay was 20 HIV-RNA copies/mL. Plasma samples were tested for an
4
116
HBV-related marker (HBsAg) using an HBV enzyme-linked immunosorbent assay
117
(ELISA) (Siemens Healthcare Diagnosis, Malvern, PA). HCV exposure (anti-HCV)
118
was detected using an HCV enzyme-linked immunosorbent assay (ELISA) (Siemens
119
Healthcare Diagnosis, Malvern, PA).
120 121
Immunophenotyping and intracellular cytokine staining of monocytes
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The methods for immunophenotyping and intracellular cytokine staining of
123
monocytes have previously been described in detail [12]. Briefly, samples consisting of
124
one mL of peripheral fresh whole blood were collected in EDTA tubes (within 30 min
125
prior to the assay). Erythrocytes were lysed according to the manufacturer’s instructions
126
(Lyse Buffer, R&D, CA), and the cells were immunophenotyped using a panel of
127
antibodies for lineage, activation, cell adhesion surface markers and a viability dye to
128
exclude nonviable cells: LIVE/DEAD, CD8, CD19, and CD40 (clone HB14) (Life
129
Technologies, CA, USA); CD3, CD4, CD56, CD16 (clone 3G8), CD11b (clone
130
ICRF44), CD62L (clone DREG-56) and CD49d (clone gF10) (BD Biosciences, NJ,
131
USA); and CD14 (clone M5E2), HLA-DR (clone L243), CD38 (clone HB-7), and
132
CD163 (clone GHI/G1),
133
Technologies, CA, USA); CD16, CD11b, CD62L and CD49d, (BD Biosciences, NJ,
134
USA); and CD38 and CD163 (Biolegend, CA, USA) were included in each experiment.
135
In vitro assays were performed on 1.5 ml of whole blood that was collected in EDTA
136
tubes (within 30 min prior to the assay). Erythrocytes were lysed according to the
137
manufacturer’s instructions (Lyse Buffer, R&D, CA). Then, the cells were resuspended
138
in R10 media (RPMI 1640 supplemented with 10% heat-inactivated calf serum, 100
139
U/ml penicillin G, 100 μl/ml streptomycin sulfate, and 1.7 mM sodium glutamine) that
140
contained 10 U/ml DNase I (Roche Diagnostics) and rested for 1 h before use. Then,
141
cells were stimulated with 1 ng/mL LPS ultrapure (Toll-like receptor (TLR) 4 agonist),
142
20 ng/mL lipomannan from M. smegmatis (LM-MS) (Toll-like receptor (TLR) 2
143
agonist) or 0.5 μg/mL ssRNA40s/Lyovec (TLR 7 agonist) (all from InvivoGen, San
144
Diego, CA, USA) for six hours for in vitro stimulation or without stimuli for the
145
unstimulated condition, in the presence of 1 μg/ml of anti-CD28, 1 μg/ml of anti-CD49d
146
(BD Biosciences, NJ, USA), 10 μg/ml of brefeldin A (BFA) (Biolegend, CA, USA) at
147
37°C/5% CO2 for six hours. Surface staining was performed using the following
148
antibodies: LIVE/DEAD, CD8, CD14, and CD19 (Life Technologies, CA, USA); CD56
(Biolegend, CA, USA). Isotype controls for CD14 (Life
5
149
and CD16 (BD Biosciences, NJ, USA); and HLA-DR (Biolegend, CA, USA). The cells
150
were washed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) and
151
stained with the following antibodies: CD3, IL-6 (clone MQ2-6A3), IL-1α (clone AS5)
152
and TNF-α (clone MAb11) (BD Biosciences, NJ, USA) and IL-1beta (β) (clone
153
JK1B1), IL-8 (clone BHO814), and IL-10 (clone JE53-9D7), (Biolegend, CA, USA).
154
Isotype controls for CD14 (Life Technologies, CA, USA); CD16, TNF-α, IL-6, and IL-
155
1α (BD Biosciences, NJ, USA); and IL-1β, IL-8, and IL-10 (Biolegend, CA, USA) were
156
included in each experiment. Monocytes were defined as high Forward (FSC)/Side
157
scatter (SSC), and expressing HLA-DR, CD14 and/or CD16, but not CD3, CD8, CD19
158
or CD56. The cells were analyzed using a LSR Fortessa Cell Analyzer (BD
159
Biosciences, NJ, USA). A minimum of 2,000,000 total events and 20,000 monocytes
160
were recorded in each tube (Supplementary Figure 1). Data were analyzed using FlowJo
161
8.7.7 (TreeStar). We calculated the responsiveness of cells in vitro by subtracting the
162
results of the unstimulated condition.
163 164
Assay of soluble biomarkers and anti-CMV titers.
165
Serum samples were collected in serum separation tubes, and plasma samples
166
were collected in EDTA tubes. The levels of high-sensitivity CRP (hsCRP) and β2-
167
microglobulin (β2M) were determined with an immunoturbidimetric serum assay using
168
Cobas 701® (Roche Diagnostics, GmbH, Mannheim, Germany). The D-dimer levels
169
were measured with an automated latex-enhanced immunoassay using plasma samples
170
(HemosIL D-Dimer HS 500, Instrumentation Laboratory, USA). The serum and plasma
171
samples were aliquoted and stored at -20°C until subsequent analysis of the following
172
biomarkers: IL-6, IL-8, IL-10, IL-1β, and TNF-α (R&D Systems, Minneapolis, MN,
173
USA), sCD14, (Diaclone, Besançon, France) and sCD163 (Macro CD163 IQ products,
174
The Netherlands). Additionally, anti-CMV IgG titers were assayed in the sera using a
175
cytomegalovirus IgG enzyme-linked immunosorbent assay (GenWay, San Diego, CA).
176 177
Statistical analysis
178
Continuous variables are expressed as medians and interquartile ranges (IQR),
179
and categorical variables are expressed as percentages. The correlations between
180
continuous variables were assessed using the Spearman rank test. The Mann-Whitney U 6
181
test was used to analyze differences between unpaired groups. Statistical analyses were
182
performed using the Statistical Package for the Social Sciences software (SPSS 17.0;
183
SPSS, Chicago, IL). Prism, version 5.0 (GraphPad Software, Inc.) was used to generate
184
the graphs. We defined polyfunctionality as the percentage of monocytes that produce
185
multiple cytokines. Polyfunctionality pie charts were constructed using Pestle, version
186
1.6.2 and Spice, version 5.2 (both kindly provided by M. Roederer, NIH, Bethesda,
187
MD) [13].
7
188
RESULTS
189
Characteristics of the study subjects
190
The demographic and immunovirological characteristics of all study participants are
191
shown in Table 1. The HIV and Young groups were predominantly men, while only
192
45% of the elderly subjects were male. The median age was 83 [67-90] years for this
193
group. The absolute total monocyte counts in the HIV group were similar to the Elderly
194
counts but higher than the Young counts. We did not find differences in the frequency
195
of monocyte subsets between the groups (data not shown).Of the 60 participants, 100%
196
were CMV-seropositive. Similar higher anti-CMV-IgG titers were found in the HIV and
197
Elderly when compared to the Young group. The HIV group showed a median nadir
198
CD4 T cell count of 330 [266-375] cells/mm3, the median time since HIV+ diagnosis
199
was 6 [3-9] years and the median time since HIV-RNA was undetectable in plasma was
200
6 [3-9] years. None of HIV-infected patients had histories of prior Category C clinical
201
stage. Neither patients nor controls had prior HCV exposure (anti-HCV negative) and
202
all of them were HBsAg negative.
203
HIV-infected patients showed a more activated phenotype of monocytes than the elderly
204
Figure 1 displays the percentage of surface expression of the activation and cell
205
adhesion markers CD163, CD49d, CD62L, CD40, CD11b and CD38 in the three
206
subsets of monocytes among the groups. The expression of the scavenger receptor
207
CD163 in the HIV group was remarkably higher compared to both control groups in the
208
three subsets of monocytes (p
