HIV-1 Infection of Human Intestinal Lamina
Propria CD4 + T Cells In Vitro Is Enhanced
by Exposure to Commensal Escherichia coli
This information is current as
of June 18, 2017.
Stephanie M. Dillon, Jennifer A. Manuzak, Amanda K.
Leone, Eric J. Lee, Lisa M. Rogers, Martin D. McCarter and
Cara C. Wilson
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Copyright © 2012 by The American Association of
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J Immunol 2012; 189:885-896; Prepublished online 11 June
2012;
doi: 10.4049/jimmunol.1200681
http://www.jimmunol.org/content/189/2/885
The Journal of Immunology
HIV-1 Infection of Human Intestinal Lamina Propria CD4+
T Cells In Vitro Is Enhanced by Exposure to Commensal
Escherichia coli
Stephanie M. Dillon,* Jennifer A. Manuzak,* Amanda K. Leone,* Eric J. Lee,*
Lisa M. Rogers,* Martin D. McCarter,† and Cara C. Wilson*
O
ne of the hallmarks of progressive HIV-1 disease is
a gradual depletion of peripheral blood CD4 T cells.
However, HIV-1 infection is also characterized by 2a
significant assault to the gastrointestinal (GI) tract, resulting in
myriad structural and immunological complications. The impact of
HIV-1 on the GI tract has been observed since the early days of
research into the epidemic, when HIV-1 infection was associated
with GI abnormalities such as diarrhea, weight loss, malnutrition,
malabsorption, and villous atrophy (1). Recent studies have
highlighted the central role played by the intestinal mucosal immune system in HIV-1 pathogenesis. Studies using a pathogenic
SIV infection model demonstrated that significant and rapid depletion of intestinal CD4 T cells occurred as early as 7 d post
infection (2–4) and was associated with early epithelial barrier
dysfunction and high levels of viral replication (2, 4). Early
in vitro studies demonstrated that human lamina propria (LP) CD4
T cells were also naturally permissive to HIV-1 infection and,
*Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045; and †Department of Surgery, University of Colorado Anschutz
Medical Campus, Aurora, CO 80045
Received for publication February 27, 2012. Accepted for publication May 11, 2012.
This work was supported by National Institutes of Health Grants 1RO1 DK088663
and 5K24 AI074343.
Address correspondence and reprint requests to Dr. Cara C. Wilson, University of
Colorado Anschutz Medical Campus, Department of Medicine, Division of Infectious Diseases, Mail Stop B168, 12700 East 19th Avenue, Aurora, CO 80045. E-mail
address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CM, complete medium; DC, dendritic cell; GI,
gastrointestinal; HK, heat killed; IFC, intracellular flow cytometry; LP, lamina propria; LPMC, lamina propria mononuclear cell; MFI, mean fluorescence intensity.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200681
unlike peripheral blood CD4 T cells, required no prior stimulation
for productive infection (5). Indeed, .50% of human intestinal
CD4 T cells are depleted during acute and early HIV infection,
and significant depletion of these cells has been noted throughout
all stages of HIV-1 disease (6–9). The susceptibility of LP T cells
to HIV-1 or SIV infection at steady state likely relates to their
increased activation status and expression of HIV/SIV coreceptors such as CCR5 and a4b7 (10–14). Depletion occurs directly
through lysis of infected cells (4, 15, 16) and indirectly through
apoptosis of infected and uninfected CD4 T cells (15). With respect to HIV infection, higher HIV viral DNA and RNA levels
were observed within GI tract CD4 T cells compared with peripheral blood CD4 T cells from subjects during acute and early
infection, with infection detected in both activated and nonactivated mucosal CD4 T cells (9). In a recent study, d’Ettorre et al.
(17) demonstrated that in chronically HIV-infected, untreated
donors, HIV DNA load was greater in the gut mucosa than in
peripheral blood.
Human Th17 cells are a subset of CD4 T cells that have been
shown to produce IL-17A, IL-17F, IL-22, and IL-26 (18) and to
play an important part both in mucosal defense against extracellular bacterial and fungal pathogens and in epithelial barrier
maintenance and regeneration (19). Thus, the loss of these cells
would be expected to have an impact on both intestinal homeostasis and immunity. A number of recent studies have highlighted
that the specific depletion of Th17 cells is associated with disease
progression in both SIV and HIV infections (20–24). Indeed, the
preservation of Th17 cells during chronic SIV infection of sooty
mangabeys has been associated with the nonpathogenic phenotype of this natural host of SIV (20).
HIV/SIV-associated epithelial barrier dysfunction and CD4
T cell depletion, in particular Th17 cell depletion, may contribute to
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Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may
contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated T cell responses in the context of ongoing viral replication that occurs
early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal
human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th
cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1Bal. Addition of heatkilled E. coli to HIV-1–exposed LPMCs resulted in increases in HIV-1 replication, CD4 T cell activation and infection, and IL-17
and IFN-g production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 T cell infection. E. coli
exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells
in HIV-1–exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E.
coli-associated enhancement of infection was dependent on the presence of CD11c+ LP dendritic cells and, in part, on MHC class
II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1
pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells. The Journal of
Immunology, 2012, 189: 885–896.
886
COMMENSAL E. coli ENHANCES HIV INFECTION OF GUT Th17 CELLS
Materials and Methods
Tissue samples and preparation of LPMCs
Human intestinal tissue samples (n = 10 jejunums, n = 11 colons) obtained
from patients undergoing elective abdominal surgery represent otherwise
discarded tissue and were considered macroscopically normal, as previously described (32, 33). All patients undergoing surgery signed a release
to allow the unrestricted use of discarded tissues for research purposes,
and all protected patient information was de-identified to the laboratory
investigators. This research was reviewed by the Colorado Multiple Institutional Review Board at the University of Colorado Anschutz Medical
Campus and was granted exempt research status. LPMCs were isolated
from tissue samples, and released LPMCs were cryopreserved and stored
in liquid nitrogen, as detailed elsewhere (32, 33). Within all LPMC samples (n = 21), the median percentage of total viable cells was 75.5% (range
30.6–96.6%), and the median percentage of viable CD45+ LPMCs was
86.6% (range 54.5–97.7%), as assessed using flow cytometry staining
protocols. Percentages of T cells as a fraction of viable CD45+ LPMCs
were evaluated in the majority of samples: CD3+ T cells (76.4%, 53.7–
95.2%; n = 20), CD4+ T cells (61.0%, 43.4–75.2%; n = 18), and CD8+
T cells (12.2%, 4.8–41.4%).
Preparation of PBMCs
Peripheral blood samples were collected from healthy adults, selfidentifying as HIV-1 negative, who voluntarily gave written informed
consent to participate. Collection of blood samples was approved by the
Colorado Institutional Review Board at the University of Colorado
Anschutz Medical Campus. PBMCs were isolated from heparinized blood
by standard Ficoll-Hypaque (Amersham Biosciences, Piscataway, NJ)
density gradient centrifugation, as described previously (34), and were
cryopreserved and stored in liquid nitrogen, as detailed elsewhere (32, 33).
Surface and intracellular flow cytometry staining assays
Standard flow cytometry staining protocols for surface markers and for
intracellular cytokine or HIV-1 p24 expression are detailed elsewhere (32,
33, 35). LP T cells were identified using CD45 (PerCp-Cy5.5; eBioscience,
San Diego, CA), CD3 (FITC; BD Biosciences, San Jose, CA; and PETexas Red, ECD; Beckman Coulter, Fullerton, CA), CD4 (APC, APC-Cy7,
AF700; all BD Biosciences), and CD8 (APC, FITC; both BD Biosciences;
and AF405, Invitrogen, Carlsbad, CA). PE-Cy5 CD38 and a matched
isotype control (BD Biosciences) were used to evaluate T cell activation.
Intracellular cytokines were detected using the following: V450 IL-17 and
AF700 IFN-g with V450 mouse IgG1 and AF700 mouse IgG1 used as
isotype controls (all BD Biosciences). Intracellular HIV-1 p24 was
detected using PE (RD1) KC57 (specific for the 55-, 39-, 33-, and 24-kDa
proteins of the core Ags of HIV-1) and PE (RD1) mouse IgG1 as a
matched isotype control (both Beckman Coulter). To evaluate CD11c and
CD19 percentages in total LPMCs and in CD11c/CD19-depleted LPMCs,
the following Abs were used: PerCp-Cy5.5 CD45, PE-Cy5 CD11c, and
APC-H7 CD19 (both from BD Biosciences). In all staining protocols,
a Live/Dead Fixable Dead Cell Stain (Invitrogen) was included. At the
completion of the staining procedure, LPMCs were resuspended in 1%
paraformaldehyde (Sigma-Aldrich, St. Louis, MO) prior to acquisition on
an LSRII Flow Cytometer (BD Biosciences).
Commensal heat-killed E. coli and Bacteroides fragilis stocks
and CCR5-tropic HIV-1Bal stock
E. coli stocks (no. 25922; ATCC, Manassas, VA) were expanded overnight in
RPMI 1640 (Invitrogen) + 10% FBS (Sigma-Aldrich) at 37˚C, 5% CO2, or
were plated on Brain Heart Infusion agar (BD Diagnostics, Sparks, MD) and
incubated at 37˚C for 1 d. B. fragilis stocks (no. 25285; ATCC) were expanded by culturing on Brucella plates (BD Diagnostics) and incubated at
37˚C for 2 d in anaerobic conditions using a BD GasPack EZ Anaerobe
Pouch System (BD Diagnostics). After expansion, bacteria were heat killed
(HK) at 56˚C for 2 h, washed, and resuspended at 3 3 109 bacteria per
milliliter in Dulbecco’s PBS and stored in single-use aliquots at 220˚C.
To prepare HIV-1 viral stocks for use in the in vitro assays, PBMCs were
resuspended at 2 3 106 cells per milliliter in RPMI 1640 + 1% penicillin/
streptomycin/L-glutamine (Sigma-Aldrich) + 10% human AB serum
(complete medium [CM]) (Gemini Bio-Products, West Sacramento, CA)
and stimulated with 5 mg/ml PHA (Sigma-Aldrich) for 3 d at 37˚C, 5%
CO2. PHA-blasted PBMCs were collected and resuspended at 1 3 106cells
per milliliter in CM + 10 U/ml rIL-2 (Roche, Indianapolis, IN) and cultured with CCR5-tropic HIV-1Bal (catalog no. 510, National Institutes of
Health AIDS Research and Reference Reagent Program, Germantown,
MD) for 7 d at 37˚C, 5% CO2. Fresh PHA-blasted PBMCs, resuspended at
1 3 106cells per milliliter in CM + 10 U/ml rIL-2 were added at day 7 as
additional “feeder cells.” At day 14 after initial viral inoculation, supernatants were collected and centrifuged at 1400 rpm for 10 min to remove
cells and cellular debris. CCR5-tropic HIV-1Bal supernatants were frozen
in single-use aliquots at 280˚C. HIV-p24 content of these viral stocks was
determined using the HIV-1 p24 ELISA (PerkinElmer, Waltham, MA), and
viral stock concentrations ranged from 0.11 to 0.26 mg/ml.
Mitogenic stimulation of LPMCs
LPMCs were resuspended at 1 3 106 cells per milliliter in CM + 500 mg/ml
piperacillin/tazobactam (Wyeth, Madison, NY) 6 250 mg/ml amphotericin
B (Invitrogen) and stimulated with 100 ng/ml PMA (Sigma-Aldrich) and 1
mg/ml ionomycin (Sigma-Aldrich) in the presence of 1 mg/ml brefeldin A
(Golgi Plug; BD Biosciences) for 5 h at 37˚C, 5% CO2. LPMCs were
collected and intracellular percentages of IFN-g– and IL-17–producing
cells determined by intracellular flow cytometry (IFC) assay.
In vitro infection assays
LPMCs or PBMCs were resuspended at 1 3 106 cells per milliliter in CM +
500 mg/ml piperacillin/tazobactam and cultured for 15–20 h with 0.08–0.2
mg p24 per milliliter of CCR5-tropic HIV-1Bal. LPMCs or PBMCs were
washed in warm Dulbecco’s PBS and resuspended at 1 3 106 cells/ml in
CM + 500 mg/ml piperacillin/tazobactam and cultured with or without
HK bacteria at 5 bacteria: 1 LPMC or PBMC in 48-well plates for 2–3 d at
37˚C, 5% CO2. In some assays, ultrapure LPS derived from commensal
E. coli strain K12 (10 mg/ml; InvivoGen, San Diego, CA) was added to
HIV-1Bal–exposed LPMCs in 96-well plates. To block MHC class II presentation, LEAF Purified anti-human HLA-DR (clone L243, 10–20 mg/ml;
BioLegend, San Diego, CA) was added to LPMCs in 96-well plates 30 min
prior to the addition of E. coli and again up to 24 h later. LEAF Purified
mouse IgG2a isotype (BioLegend) was used as the control Ab.
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reduced protection from microbial products translocating from the
lumen into the LP and into the systemic circulation. Using a model
of intestinal inoculation of Salmonella typhimurium during acute
SIV infection of rhesus macaques, Raffatellu et al. (25) observed
depletion of Th17 cells, a decrease in epithelial barrier integrity,
and the dissemination of S. typhimurium. In a recent study, Estes
et al. (26) demonstrated the presence of not only microbial products such as LPS, but also Escherichia coli, in colonic LP and in
lymph nodes of chronically infected rhesus macaques, providing
more direct evidence for the occurrence of gut-associated microbial translocation in SIV infection. Indirect evidence for this
phenomenon occurring in HIV-1 infection has also been demonstrated. Increased levels of microbial products were observed in
the circulation of HIV-1–infected individuals and were found to be
associated with T cell activation (27, 28).
Epithelial barrier dysfunction is known to occur early in SIV and
HIV disease (2, 29–31) in association with microbial translocation
(26). However, the mechanisms by which translocated microbes
induce mucosal immune dysfunction and impact viral replication remain poorly understood. We have previously identified significant
frequencies of human LP effector CD4 T cells that produced IFNg and IL-17 in response to commensal bacteria in normal human
intestinal tissue (32). The in vitro expansion of these bacteriareactive T cells was dependent upon the presence of a subset of
LP dendritic cells (DCs). We hypothesized that in the setting of
HIV infection and a “leaky” gut barrier, LP mononuclear cells
(LPMCs) would have increased exposure to bacteria and bacterial
products. This exposure, in turn, would lead to the activation of
bacteria-reactive T cells, increasing their permissiveness to HIV
infection and replication. Using an in vitro assay that mimics the
early interactions between HIV-1, commensal bacteria, and primary LPMCs, we present evidence to suggest not only that IL-17–
producing intestinal CD4 T cells are preferentially infected, but
also that productive infection is further enhanced in the presence
of commensal E. coli. These results provide a mechanism for how
microbial translocation may contribute to increased viral replication and CD4 T cell depletion in mucosal tissue.
The Journal of Immunology
At the completion of the culture period, culture supernatants were collected and frozen at 220˚C for subsequent assessment of secreted IFN-g,
IL-17, and HIV-1 p24. LPMCs or PBMCs were collected and either immediately assessed for surface expression of CD38 and/or intracellular
expression of HIV-1 p24 by flow cytometry or underwent mitogenic
stimulation, with intracellular HIV-1 p24 percentages within cytokineproducing cells determined by IFC assay.
T cell proliferation assays
LPMCs were prelabeled with 5 mM CFSE (Invitrogen) in HBSS for 15 min
at 37˚C and washed with CM. CFSE-labeled LPMCs were resuspended
at 1 3 106 cells/ml in CM and cultured with or without HK E. coli (5 E.
coli:1 LPMC) for 7 d at 37˚C, 5% CO2. PHA (5 mg/ml; Remmel, Lenexa,
KS) was used as a positive control, and all samples had PHA-induced
CD4+ T cell proliferation .50% (range: 52.7–98.8%; data not shown).
To determine proliferation of cytokine-producing cells, total LPMCs were
then collected after 7 d of in vitro culture, underwent mitogenic stimulation, and were assessed for intracellular cytokine expression by IFC assay.
Identification of cytokine-producing bacteria-reactive LP CD4
T cells
Depletion of CD11c+ and CD19+ LP cells
LPMCs were stained with biotinylated CD11c (Miltenyi Biotec, Auburn,
CA) 6 biotinylated CD19 (eBioscience). Control LPMCs were resuspended in buffer only. In all cases, FcR blocking reagent (Miltenyi Biotec)
was added. Biotinylated Ab-bound cells were incubated with 32 3 105
Biotin Binder Dynabeads (Invitrogen) per 1 3 106 LPMCs for 30 min at
4˚C under constant rotation. Dynabead-bound LPMCs were magnetically
removed following the manufacturer’s recommended protocol. CD11c+
LPMCs were depleted by a median 80.3% (range: 71.4–93.8%), and
CD19+ LPMCs were depleted by 91.4% (82.3–95.6%), compared with
total viable CD45+ LPMCs (n = 7). Following depletion of CD11c+ and
CD19+ LPMCs, the percentage of CD3+ T cells increased from 78.7%
(range: 60.9–88.8%) to 85.2% (range: 69.6–93.9%) of viable CD45 +
LPMCs. For LPMC samples depleted of CD11c only (n = 3), CD11c+
LPMCs were depleted by 85.5% (range: 82.6–95%).
LPMCs were either labeled with CFSE and cultured with or without HK
E. coli (5 E .coli:1 LPMC), as described above, for 5 d or were pre-exposed
to HIV-1Bal (0.08 mg p24 per milliliter) for 19–20 h, washed, and cultured
with or without HK E. coli (5 E. coli:1 LPMC) for an additional 3 d, as
described above.
Flow cytometric acquisition and analysis
For all in vitro assays, a lymphocyte gate was established using forward
scatter-area and side scatter-area properties within viable LPMCs.
Doublets were then excluded using a forward scatter-height versus forward scatter-width dot plot. A total CD3 gate was established from within
this population of total LPMCs. In all assays, except for the in vitro
infection assays, CD4 T cells were then gated from within the CD3
gate. In our initial in vitro infection assays, we observed significant
downregulation of CD4 on CD3+ LP T cells in infected LPMC cultures
(data not shown); therefore, a CD82 gate was established to include all
CD4 T cells for these assays.
To evaluate expression of CD38, intracellular p24, and intracellular
cytokines, matched isotype Abs were used to establish background staining
and then specific expression was determined by removing the background
staining. Specific proliferation in response to E. coli was ascertained by
establishing a CFSElo gate based on the unstimulated condition.
All flow cytometry data were acquired on an LSRII Flow Cytometer. To
control for the accuracy and precision of measurements taken over the
course of the study, routine quality control using the Cytometer Setup and
Tracking feature within the BD FACSDiva software version 6.1.2 (BD
Biosciences) was performed daily as previously detailed (35).
IFN-g, IL-17, and HIV-1 p24 ELISAs
The recommended manufacturer’s protocols were followed for IFN-g (BD
Biosciences), IL-17 (eBioscience), and HIV-1 p24 ELISAs. The lower
detection limits were 4.7 pg/ml for IFN-g, 4 pg/ml for IL-17, and 12.5 pg/
ml for HIV-1 p24.
Statistical analysis
Nonparametric statistics were used. The Wilcoxon matched-pairs signed rank
test was used to evaluate paired data. The Friedman test was used for matchedpaired comparisons across multiple groups, with a multiple Dunn comparison
test performed when the overall p value was , 0.05. When nonparametric
tests failed owing to very small sample sizes, parametric tests were used.
A paired t test was used to evaluate paired data. The repeated-measures
ANOVA was used for matched-paired comparisons across multiple groups,
with a Bonferroni multiple comparison test performed when the overall p
value was , 0.05. All statistical analyses were done using GraphPad Prism
version 5.00 for Windows (GraphPad Software, San Diego, CA).
Results
Exposure to commensal E. coli increases HIV-1 replication and
infection of LP T cells in vitro
In agreement with previous studies (5), we observed productive
infection of LPMCs exposed to HIV-1Bal in the absence of exogenous stimulation (Fig. 1). LPMCs pre-exposed to HIV-1Bal +
E. coli significantly produced higher levels of HIV-1 p24 compared with those with virus only (Fig. 1A). To determine if increased viral replication and infection in the presence of
commensal E. coli was specific to intestinal T cells, we obtained
PBMCs from unmatched donors and exposed them to HIV-1Bal
and E. coli, using the same in vitro infection protocol (n = 9).
Evaluation of HIV-p24 levels within culture supernatants failed to
demonstrate an increase in HIV-p24 levels when PBMCs were
cultured with HIV-1Bal + E. coli versus HIV-1Bal alone, and, rather,
a decrease in HIV-p24 was noted (Fig. 1B). Thus, in contrast to
HIV-1Bal + E. coli–exposed LPMCs, we did not observe a similar
enhancing effect of bacteria on HIV replication in HIV-exposed
PBMCs, demonstrating the gut specificity of this process.
To enumerate the percentage of productively infected LP CD4
T cells, we assessed intracellular p24 expression, using flow
cytometry. We observed significant downregulation of CD4 on
CD3+ LP T cells in infected LPMC cultures (data not shown);
therefore, we assessed expression of intracellular HIV-1 p24 in
CD3+ CD82 T cells (Fig. 1C). Addition of E. coli to HIV-1Bal–
exposed LPMCs also resulted in significantly higher percentages
of CD82 T cells expressing intracellular HIV-1 p24 than in infected cultures without bacteria (Fig. 1D).
Exposure to HIV-1Bal and commensal E. coli increases T cell
activation and cytokine production in vitro
The T cell activation state is closely linked to the efficiency of
HIV-1 infection and replication; therefore, we also assessed CD38
expression, an indicator of T cell activation (14), on T cells in
LPMC cultures stimulated in vitro, as described above, using flow
cytometry. The addition of E. coli to HIV-1–exposed LPMCs significantly increased CD38 expression on CD82 LP T cells above
that observed in HIV-1Bal–only cultures (Fig. 2A). CD38 expression on CD8+ T cells exposed to HIV-1Bal and E. coli [median
mean fluorescence intensity (MFI): 547; range: 404–1736] was
also greater than that on CD8+ T cells exposed to HIV-1Bal only
(MFI: 577; range: 317–1101), although this difference failed to
reach statistical significance (p = 0.07). The absolute levels of
CD38 expression on CD82 T cells (MFI: 886; range: 745–3011)
were higher in response to HIV-1Bal + E. coli, than levels observed
on CD8+ T cells (MFI: 547; range: 404–1736; p = 0.004).
Low levels of IFN-g and IL-17 were detected in culture supernatants from LPMCs exposed to HIV-1Bal only (Fig. 2B, 2C).
However, both IFN-g and IL-17 significantly increased in the
presence of E. coli (Fig. 2B, 2C). In a subset of experiments,
LPMCs were pre-exposed to HIV-1Bal and cultured with a range of
doses of E. coli for 2–3 d. As the ratio of E. coli to LPMCs decreased from 5:1 to 0.2:1, a trend toward decreasing levels of
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LPMCs were resuspended at 1 3 106 cells per milliliter in CM and cultured
for 4 h with E. coli or B. fragilis (5 bacteria:1 LPMC) prior to the addition
of 1 mg/ml brefeldin A. After an additional 14–16 h, LPMCs were collected and frequencies of CD4+ T cells producing IFN-g or IL-17 were
determined by IFC assay.
887
888
COMMENSAL E. coli ENHANCES HIV INFECTION OF GUT Th17 CELLS
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FIGURE 1. Exposure to commensal E. coli increases HIV-1 replication and infection of LP T cells in vitro. LPMCs or PBMCs were pre-exposed to
CCR5-tropic HIV-1Bal and cultured with or without HK E. coli for 2–3 additional days. (A and B) p24 levels within (A) LPMCs (n = 7 jejunums, d; n = 5
colons, s) and (B) PBMC culture supernatants (n = 9). (C) Gating strategy used to identify intracellular HIV-1 p24+ LP CD4 T cells. An initial viable
lymphocyte gate was established and doublets excluded using a forward scatter height versus forward scatter width gate (profiles not shown). To account for
the downregulation of CD4 after exposure to HIV-1Bal in vitro, LP CD4 T cells were identified as CD3+ CD82 (left plot). The net expression of HIV-1 p24
within LP CD3+ CD82 T cells exposed to HIV-1Bal only (upper panel) or to HIV-1Bal and E. coli (lower panel) was established using an isotype control
(Mouse IgG1). (D) Cumulative net percentages of intracellular HIV-1 p24+ CD3+ CD82 T cells within LPMC cultures (n = 7 jejunums, d; n = 3 colons, s).
Statistical significance was determined using the Wilcoxon matched-pairs signed rank test.
CD38 expression on CD82 T cells and decreasing IFN-g and IL17 production was observed (data not shown), showing bacterial
dose-dependent effects on CD82 T cell activation.
Statistically higher levels of IFN-g and IL-17, and a trend toward higher levels of HIV-1 p24, were detected in culture
supernatants from jejunal LPMCs compared with colon LPMCs
stimulated with E. coli after pre-exposure to HIV-1Bal (Supplemental Table I). However, irrespective of anatomical location, the
amount of cytokines produced and HIV-1 p24 levels were always
greater following combined HIV-1Bal and bacterial exposure than
those observed in HIV-1Bal only.
Purified LPS from commensal E. coli does not increase HIV-1
infection within LPMCs
Increased levels of LPS in the blood of HIV-infected individuals
has been associated with systemic immune activation (27), and
increased LPS levels were observed in the colon of SIV-infected
rhesus macaques during the late acute stage of SIV infection (26).
The Journal of Immunology
889
1Bal. This finding was likely related to the lack of T cell activation
and expansion in response to LPS, as indicated by minimal induction of IFN-g (Fig. 3C) or IL-17 (Fig. 3D).
Expansion of IFN-g– and IL-17–producing LP CD4+ T cells in
response to commensal E. coli stimulation in vitro
Infection frequencies of IFN-g– and IL-17–producing T cell
subsets increase in response to exposure to HIV-1Bal and
commensal E. coli in vitro
FIGURE 2. Exposure to HIV-1Bal and commensal E. coli increases T
cell activation and cytokine production in vitro. LPMCs were pre-exposed
to CCR5-tropic HIV-1Bal and cultured with or without HK E. coli for 2–3
additional days. (A) Cumulative data showing MFI of CD38 expression on
CD3+ CD82 T cells minus isotype control values (net MFI) (n = 6 jejunums, d; n = 3 colons, s). IFN-g (B) and IL-17 (C) within culture
supernatants (n = 7 jejunums, d; n = 5 colons, s). Statistical significance
was determined using the Wilcoxon matched-pairs signed rank test.
Therefore, we wished to determine whether exposure of LPMCs to
purified commensal E. coli-derived LPS would lead to increased
infection of LP CD4 T cells, as was observed with whole E. coli.
As in previous assays, the addition of whole E. coli increased HIV1 infection and replication within CD82 T cells and production of
IFN-g and IL-17 within total LPMCs (Fig. 3A–D). However, the
addition of purified E. coli-derived LPS did not increase levels of
HIV-1 p24 (Fig. 3A) or frequencies of HIV-1 p24+ CD82 T cells
(Fig. 3B) above those observed in cultures exposed only to HIV-
We next assessed whether expansion of IL-17+ CD4+ T cells in response to E. coli (Fig. 4) corresponded to greater levels of productive
infection within this population of cytokine-producing T cells. Although intracellular HIV-1 p24 was detected in both IFN-g and IL17–producing CD82 T cells following HIV-1Bal infection, a significantly higher percentage IL-17–producing T cells expressed HIV-1
p24, indicating an inherent preference of HIV-1Bal for LP Th17 cells
(Fig. 5A). The percentage of infected IL-17+ and IFN-g+ CD82
T cells increased post infection with HIV-1Bal in the presence of E.
coli, with the fraction of infected IL-17+ CD82 T cells remaining
statistically higher than IFN-g+ CD82 T cells (Fig. 5A). We next
evaluated intracellular HIV-1 p24 expression within each cytokineproducing T cell subset in response to HIV-1Bal + E. coli, using the
gating shown in Fig. 5B. The highest median percentage of HIV-1
p24+ cells was observed in the IL-17+IFN-g+ CD82 T cell population
and the next highest percentage observed within CD82 T cells producing IL-17 only (Fig. 5C). Elevated percentages of HIV-1 p24+
cells were observed in IFN-g+ IL-172 CD82 T cells compared with
non–cytokine-producing T cells, but these were lower than those
noted for either of the IL-17–producing populations (Fig. 5C).
The ability of specific bacteria to enhance HIV-1 infection is
related to frequencies of bacteria-reactive CD4 T cells in
LPMCs
To determine whether enhancement of HIV-1 replication in LPMCs
was unique to E. coli or was generalizable to other enteric bac-
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High percentages of CD4+ T cells capable of producing IFN-g
were measured in primary LPMCs (median: 65.2% of CD4+
T cells; range: 33.6–73.2%) after 5-h stimulation with PMA/
ionomycin, with lower frequencies found of CD4+ T cells capable of producing either IL-17 alone (median: 1.9%; range: 1.4–
10.6%) or coproducing both IL-17 and IFN-g (median: 1.0%;
range: 0.1–5.6%) (Supplemental Fig. 1).
To determine the proliferative capacity of the cytokine+ CD4+
T cell subsets in response to bacteria, LPMCs were initially labeled
with CFSE, infected with HIV-1, then cultured with bacteria. E. coliinduced proliferation was detectable by flow cytometry after 5–7 d,
but viability of HIV-exposed LPMCs substantially decreased after
5 d in culture. Thus, the proliferative capacity of specific cytokine+
CD4+ T cell subsets in response to E. coli was subsequently determined after 7 d of stimulation with E. coli in the absence of HIV-1Bal.
In agreement with our previous study (32), we observed a significant
increase in the percentage of CFSElo, proliferating CD4+ T cells in
LPMCs cultured in the presence of E. coli, compared with unstimulated cultures (Fig. 4A). An example of the gating strategy used to
evaluate proliferation of the cytokine+ CD4+ T cell subsets is shown
in Fig. 4B. A hierarchy of proliferation within the specific cytokine
populations in response to E. coli was observed (Fig. 4C). The
highest fraction of proliferating cells was observed among the IL-17/
IFN-g–coproducing CD4+ T cells, with lower percentages in the
IL-17+ IFN-g2 CD4+ T cell population and the lowest fraction occurring within IFN-g+ IL-172 CD4+ T cells (Fig. 4C). A trend toward greater proliferation of all IL-17–producing CD4 T cells than of
total IFN-g–producing T cells was noted (p = 0.06; data not shown).
890
COMMENSAL E. coli ENHANCES HIV INFECTION OF GUT Th17 CELLS
teria, we compared T cell infection frequencies and cytokine
responses to E. coli versus those to B. fragilis, another Gramnegative commensal organism. The Bacteroides group has been
shown to be an abundant taxa in intestinal mucosal biopsies (36)
and therefore might be likely to translocate into the intestinal LP
during epithelial barrier breakdown. However, when LPMCs were
pre-exposed to HIV-1Bal and then cultured with either E. coli or B.
fragilis, B. fragilis failed to induce the increases in HIV-1 replication, T cell activation and infection, and cytokine production
that were observed following E. coli exposure (Fig. 6A–D). To
determine whether these differential responses were potentially
related to differences in frequencies of resident bacteria-reactive
LP CD4+ T cells, the frequencies of IFN-g– and IL-17–producing
cells responding to each bacterial species were measured in normal LPMCs, using intracellular cytokine staining, as described
(32). LPMCs contained significantly higher frequencies of IFNg– and IL-17–producing CD4+ T cells that recognized E. coli than
those that recognized B. fragilis (Fig. 6E).
Commensal E. coli-associated proliferation, cytokine
production, and enhancement of infection of LP CD4+ T cells
require the presence of CD11c+ LP cells and are, in part, MHC
class II restricted
We previously showed that bacteria-specific LP T cell proliferation
was dependent on the presence of a subset of LP DCs (32). We
wished to determine if the E. coli-associated increase in HIV-1
infection of LP T cells observed in our current in vitro assays was
also dependent on the presence of APCs or, rather, represented
a direct effect of E. coli on T cells. To enrich for T cells, LPMCs
were depleted of CD11c+ (DCs) and CD19+ (B cells) cells, using
magnetic cell separation techniques. Depletion of DCs and B cells
trended toward reduced proliferation of E. coli-reactive LP T cells
relative to that observed in total LPMC cultures (p = 0.07; Fig.
7A), with an overall mean decrease of 71.5% (SEM: 8.5%; n = 4).
In tandem with the decreased proliferation in response to E. coli,
lower levels of IFN-g and IL-17 were also observed within the
T cell-enriched LPMC cultures, compared with levels detected
within total LPMC cultures following E. coli stimulation (Fig.
7B), although this did not reach statistical significance for IFN-g.
This observed decrease in cytokine production equated to a mean
decrease of 82.5% (SEM: 5.6%) in IFN-g and 66.8% (SEM:
15.1%) in IL-17 levels. Thus, the E. coli-induced expansion and
cytokine production were not occurring primarily through direct
stimulation of T cells and implicated a role for CD19+ and/or
CD11c+ LP cells.
We next investigated whether the requirement for CD19+ and
CD11c+ LP cells in the expansion of E. coli-reactive T cells also
impacted bacteria-associated enhancement of HIV-1 infection of
LP T cells. To further define the role of DCs, we also depleted
LPMCs of CD11c+ DCs only. Minimal changes in the levels of
HIV-1 p24 from HIV-1–exposed LPMCs cultured in the absence of
E. coli were detected when CD11c and CD19 LP cells were depleted (Fig. 7C). However, we observed a decrease in the bacterial
enhancement of infection in CD11c/CD19-depleted cultures (Fig.
7C), with levels of supernatant HIV-1 p24 decreasing by 46.9%
(SEM: 4.5%). Comparison with CD11c-depleted cultures showed
similar reductions in infection levels in the presence of E. coli (Fig.
7C), with HIV-1 p24 levels decreasing by 49.4% (SEM: 9.3%),
suggesting that depletion of DCs rather than B cells was responsible
for the majority of effects observed. Notably, mean levels of HIV-1
p24 detected in CD11c/CD19- and CD11c-depleted cultures stimulated with E. coli were similar (CD11c/CD19-depleted: 2858
pg/ml; CD11c-depleted: 2908 pg/ml) to levels observed within culture supernatants from total LPMCs exposed only to HIV-1Bal
(2417 pg/ml), further highlighting the role for LP DCs in driving the
E. coli-associated enhancement of HIV-1 infection of LP T cells.
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FIGURE 3. Exposure to HIV-1Bal and commensal E. coli-derived LPS does not increase HIV-1 replication and infection of LP T cells or IFN-g and IL-17
cytokine production in vitro. LPMCs were pre-exposed to CCR5-tropic HIV-1Bal and cultured with or without HK E. coli or purified LPS derived from
commensal E. coli (Strain K12) for 2–3 additional days. (A–D) Cumulative data showing (A) p24 levels within culture supernatants, (B) net percentages of
intracellular HIV-1 p24+ CD3+ CD82 T cells within LPMC cultures, and (C) IFN-g and (D) IL-17 levels within culture supernatants. Each symbol represents an individual sample (n = 4 jejunums). Statistical significance was determined using the repeated measures ANOVA (overall p value) with
a Bonferroni multiple comparison test. **p , 0.01, ***p , 0.001.
The Journal of Immunology
891
in the absence of E. coli (Fig. 7D). However, significantly reduced
levels of HIV-1 p24 were detected in culture supernatants after
stimulation of HIV-1Bal–exposed LPMCs to E. coli in the presence
of the HLA-DR blocking Ab (Fig. 7D), with levels in the presence
of anti–HLA-DR Ab decreasing by 56.2% (SEM: 9.5%) relative to
levels detected in the presence of the isotype control. Taken together, these results show that the bacterial enhancement of HIV
replication and infection within LP CD4 T cells does not occur
through direct stimulation of T cells but, rather, requires the
presence of CD11c+ DCs and is partially MHC class II restricted.
Discussion
We have previously shown that expansion of E. coli-specific LP
T cells was partially dependent on HLA-DR molecules (32). To
further delineate potential mechanisms behind the current observations that LP DCs were required for the E. coli-associated enhancement of LP T cell infection, we evaluated levels of infection
in the presence of an HLA-DR blocking Ab. Minimal effect of
HLA-DR blocking on the levels of HIV-1 infection was observed
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FIGURE 4. E. coli induces the expansion of LP Th17 cells. CFSE-labeled LPMCs were cultured for 7 d with or without heat-killed E. coli
followed by a 5-h mitogenic stimulation. (A) Percentages of CFSElo cells
within CD3+ CD4+ T cells in unstimulated cultures and within cultures
stimulated with E. coli (n = 3 jejunums, d; n = 3 colons, s). Statistical
significance was determined using the Wilcoxon matched-pairs signed
rank test. (B) Gating strategy used to determine the percentages of proliferating cells within each cytokine+ T cell population in response to E.
coli. CD3+ CD4+ T cells were gated from within a viable lymphocyte gate
with a doublet discrimination gate applied (profiles not shown). IFN-g–
and IL-17–expressing CD4+ T cells were determined using isotypes. The
CFSElo gate within IL-17+ IFN-g+, IL-17+ IFN-g–, and IFN-g+ IL-172
CD4+ T cells was established from unstimulated cultures. (C) Cumulative
results from 6 samples (n = 3 jejunums, d; n = 3 colons, s). Statistical
significance determined using the Friedman test with a Multiple Dunn
comparison test. **p , 0.01.
In recent years, Th17 cell dynamics during HIV infection have been
extensively studied, and it is well established that significant depletion of these cells occurs both in peripheral blood (20, 37–40)
and in the intestinal mucosa (20, 22, 24, 41). Decreases in intestinal Th17 cells during HIV infection may be a factor contributing
to the translocation of bacteria and bacterial products into the LP
(26) and into the systemic circulation of HIV-1–infected individuals (27, 28). Indeed, microbial translocation occurs throughout
the course of HIV disease and has been associated with systemic
immune activation, a predictor of disease progression (27, 28, 42,
43). A link between microbial translocation and poor clinical
outcomes, such as cardiovascular disease, dementia, and mortality,
has also been suggested (44–46).
The mechanisms underlying the depletion of Th17 cells are being actively investigated. HIV infection of peripheral blood Th17
cells has previously been demonstrated both in vivo (20) and
in vitro (37, 39, 47). To evaluate whether Th17 cells were preferentially infected in vivo, Brenchley et al. (20) stimulated PBMCs
from HIV-1–infected donors with anti-CD3 and observed no
preferential infection of peripheral blood Th1 or Th17 cells. In
chronically SIV-infected rhesus macaques, spleen IFN-g+ and
IL-17+ T cells had similar levels of SIV DNA (21). In contrast,
Gosselin et al. (37) identified IL-17–producing T cells by surface
expression of CCR6 and observed higher levels of integrated
DNA within CCR6+ memory T cells relative to CCR62 memory
T cells isolated from untreated, viremic HIV-infected individuals,
suggesting that preferential infection of Th17 cells in vivo may in
fact occur. Further, in vitro infection studies have suggested that
peripheral blood Th17 cells were more permissive to HIV infection than were Th1 cells (37, 47). However, studies to address the
mechanisms responsible for HIV-associated depletion of intestinal
Th17 cells have been hampered by the small numbers of LPMCs
typically obtained from clinical gut biopsies.
In the current study, we evaluated how HIV-1 replication and
infection of resident LP T cells were influenced by exposure of
LPMCs to commensal bacteria, to better understand the mechanisms by which microbial translocation might contribute to HIV
pathogenesis at the mucosal level, particularly during early infection, when epithelial barrier breakdown, increased translocation
of intestinal microbes, and increased viral replication are known
to occur. Using an in vitro system modeling these early interactions between LPMC, HIV-1, and translocated whole bacteria, we
demonstrate that exposure to commensal E. coli increases the
activation and expansion of resident E. coli-reactive T cells
resulting in increased HIV-1 replication and infection of IFNg– and IL-17–producing CD4 T cells. A hierarchy of infection
was noted, with the greatest fraction of HIV-1 p24-expressing
cells observed within the IL-17+ IFN-g+ population, followed by
cells producing only IL-17, and finally those that produced IFN-g
only. This infectivity profile paralleled the proliferation profile of
Th1 and Th17 subsets in response to E. coli. Furthermore, the
ability of E. coli to enhance HIV-1 replication in LPMCs was
892
COMMENSAL E. coli ENHANCES HIV INFECTION OF GUT Th17 CELLS
linked to the frequency of resident LP CD4 T cells able to recognize that specific bacterial species, as HIV-1 replication was not
enhanced in response to B. fragilis, a commensal organism recognized by few LP T cells.
It is well established that activated/memory T cells, including
HIV-1–specific CD4 T cells, are preferentially infected by HIV-1
(14, 48–51). Studies have also implicated pathogenic bacteria in
enhancing HIV-1 replication. Bacterial vaginosis-associated microflora were shown to enhance HIV replication within a promonocytic cell line chronically infected with HIV-1 provirus (52);
and in HIV-1–infected subjects with active tuberculosis, higher
levels of HIV gag DNA were observed in Mycobacterium tuberculosis-specific peripheral CD4 T cells than in total memory CD4
T cells (53). In addition, this group demonstrated higher HIV infection rates within M. tuberculosis-specific CD4 T cells following
in vitro mitogenic stimulation (53). Within the intestine, the activated phenotype of LP T cells has been linked to their natural
susceptibility to HIV infection and to their rapid subsequent de-
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FIGURE 5. Greater productive HIV-1 infection of LP Th17 cells coproducing IFN-g.
LPMCs (n = 4 jejunums, d; n = 3 colons,
s) were pre-exposed to CCR5-tropic HIV1Bal and cultured with or without HK E. coli
for 3 additional days, followed by a shortterm mitogenic stimulation. (A) Net percentages of IL-17+ CD3+ CD82(Total IL17) or IFN-g+ CD3+ CD82 (Total IFN-g)
T cells expressing HIV-1 p24 within LPMC
cultures in response to HIV-1Bal only and
to HIV-1Bal + E. coli. Statistical significance was determined using the Wilcoxon
matched-pairs signed rank test. (B) Gating
strategy to determine net HIV-1 p24 expression within CD3+ CD82 T cell cytokine6 subsets (IL-17+ IFN-g+, IL-17+ IFNg2, IFN-g+ IL-172, and IFN-g2 IL-172) in
response to HIV-1Bal + E. coli. (C) Cumulative percentages of net CD3+ CD82 cytokine+/2 HIV-1 p24+ T cells within LPMC
cultures in response to HIV-1Bal + E. coli.
Statistical significance was determined using
the Friedman test with a Multiple Dunn
comparison test for each of the cytokine+ T
cell populations relative to IFN-g2 IL-172
CD3+ CD82 T cells. *p , 0.05, ***p ,
0.001.
pletion (5, 14). The activated state of intestinal T cells may be
partially explained by the presence of commensal bacteriareactive T cells (32). Our current study expands on these previous findings by addressing the potential impact of translocating
commensal bacteria on the local immune response within the intestinal LP during HIV-1 infection. Notably, our study suggests
that the translocation of certain enteric bacteria across a leaky
epithelial barrier into the intestinal LP could increase the pool
of activated resident LP CD4 T cells, particularly Th17 cells,
resulting in increased infection rates. These in vitro findings
provide a physiological basis by which translocating microbes
might contribute to mucosal Th17 cell depletion, but this theoretical process remains to be verified as a true mechanism of
HIV-1 pathogenesis in vivo.
In studies to address the potential mechanisms behind the
bacteria-associated enhancement of HIV-1 infection and replication within LP T cells, we demonstrated that exposure of infected
LPMCs to purified LPS, a known TLR4 agonist (54), did not result
The Journal of Immunology
893
in the increases in HIV-1 replication that were observed using
whole bacteria. We previously identified MHC class II-restricted,
Th1 and Th17 bacteria-reactive T cells within the LP of normal
human intestinal tissue (32) and showed that their proliferation
was DC dependent. We now show that the ability of bacteria to
enhance HIV-1 replication in LP CD4 T cells is also dependent on
the presence of LP DCs, and is, in part, MHC class II restricted.
Taken together, these findings suggest that innate stimulation of
single bacterial TLR alone is insufficient to result in T cell activation and increased infection and that Ag presentation by DCs is
likely critical to the expansion of resident bacteria-reactive CD4
T cells necessary for increased HIV-1 replication. However, other
factors, such as cytokines, are also probably involved in the enhancement of HIV-1 replication, as MHC class II blockade only
partially inhibited this process. The bacterial Ags, either alone or
in combination, that are responsible for the observed enhancement
of infection and replication, as well as the DC-derived cytokines
or chemokines involved, remain to be determined.
One of the more intriguing observations made in our study is the
bacteria-induced selective expansion and infection of Th17 cells
that coproduce IFN-g. Although not extensively researched, this
Th17/Th1 subset has been described in other investigations
addressing human peripheral blood Th17 responses in the setting
of HIV-1 infection. IL-17/IFN-g–coproducing T cells expressed
CCR5 and were productively infected after exposure to CCR5tropic HIV-1 in vitro (37, 47), and in one study, the highest levels
of in vitro infection and depletion were noted in IL-17+ IFN-g+
CD4 T cells (47). During early HIV infection, higher frequencies
of HIV-specific IL-17+ IFN-g+ blood CD4 T cells have also been
noted in vivo (55). To the best of our knowledge, this current study
is the first to show that commensal E. coli induced expansion of
human LP IL-17+ IFN-g+ CD4 T cells, increasing the frequency of
productively infected cells when concurrently exposed to CCR5tropic HIV-1 in vitro.
In a recent study tracking IL-17–producing cells in an in vivo
mouse model, Hirota et al. (56) demonstrated that IL-23 was
necessary not only for the induction of full effector function of
Th17 cells, but also for induction of the Th1-associated transcription factor Tbet and the subsequent switch of these cells to an
IL-17+ IFN-g+ T cell phenotype. Importantly, this plasticity of IL17–producing cells appeared only during experimental autoimmune encephalomyelitis, a chronic inflammatory disease, and not
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FIGURE 6. E. coli induces greater productive infection and activation of LP T cells compared with B. fragilis. LPMCs (n = 3 jejunums, d; n = 3 colons,
s) were pre-exposed to CCR5-tropic HIV-1Bal and cultured with or without HK E. coli or B. fragilis for 3 additional days. Fold increases in (A) HIV-1 p24
levels within culture supernatants, (B) percentages of intracellular HIV-1 p24+ CD3+ CD82 T cells, and (C) CD38 expression by CD3+ CD82 T cells within
LPMC cultures exposed to HIV-1Bal and either E. coli or B. fragilis above that observed in LPMC cultures exposed to HIV-1Bal only. (D) IFN-g and IL-17
levels detected in culture supernatants within LPMC cultures exposed to HIV-1Bal and either E. coli or B. fragilis above that observed in LPMC cultures
exposed to HIV-1Bal only (net). (E) Frequencies of CD4+ T cells expressing IFN-g or IL-17 after 18–20 h of exposure to either E. coli or B. fragilis.
Statistical significance was determined using the Wilcoxon matched-pairs signed rank test.
894
COMMENSAL E. coli ENHANCES HIV INFECTION OF GUT Th17 CELLS
postinfection of the skin with Candida albicans, which resulted in
acute inflammation, rapid clearance of the fungus, and a relatively
anti-inflammatory environment. A proinflammatory environment
as a requirement for “switching” of Th17 cells to IL-17/IFN-g–
coproducing T cells has also been observed in a mouse model
of ocular inflammation (57). Thus, in the context of our HIV-1infection assay, we speculate that the expansion of these cells and
their increased infection in the presence of HIV-1 and E. coli
in vitro may result in part from the proinflammatory milieu generated. However, further detailed analysis of LP Th1/Th17 cells,
including their expression of Th1 and Th17 transcription factors
and dependence on specific cytokines induced by enteric bacteria,
is necessary to define a possible role for them in HIV-1–associated
mucosal immune dysfunction.
Why do IL-17–producing LP CD4 T cells, and in particular
those that coproduce IL-17 and IFN-g, constitute the greatest
fraction of infected cells after exposure to HIV-1 and E. coli?
Several studies have shown that intestinal Th17 cells are highly
infectable, which may be in part attributable to the high level of
baseline activation of LP CD4 T cells (14), as well as the expression of HIV coreceptors. LP CD4+ T cells express high levels
of CCR5 (5, 58), and CCR5-expressing LP Th17 cells were shown
to be preferentially depleted in HIV-infected subjects (20). In
addition, peripheral blood IL-17+ IFN-g+ CD4 T cells were shown
to highly express CCR5 (37, 47). Recently, HIV and SIV have
been demonstrated to bind the gut-homing receptor a4b7 on
peripheral blood CD4+ T cells in vitro and in vivo (10, 11), and the
a4+b7hi T cells contained a substantial proportion of IL-17–producing cells (11). Intestinal a4+b7hi CD4 T cells were also preferentially infected in the early stages of SIV infection (59). Thus,
expression of a4b7, as well as CCR5, may explain the greater
infection frequency of intestinal Th17 cells in our in vitro infection assay. In addition, peripheral blood Th17 cells were shown to
have a reduced ability to produce MIP-1a and MIP-1b, CCR5
ligands associated with blocking of HIV replication, relative to
IFN-g–producing T cells, thus providing another potential
mechanism for a greater infectivity rate of IL-17– versus IFN-g–
producing T cells (47).
Our data suggest that the intrinsic targeting of IL-17–producing
cells by HIV-1 can be further enhanced upon exposure to E. coli,
a process that appears to be dependent upon the baseline frequency of bacteria-reactive cells and their capacity to proliferate
upon bacterial exposure. In particular, the selective activation and
expansion of IL-17/IFN-g–coproducing cells in response to bacteria likely contributes to their selective targeting by HIV-1 in our
in vitro model system. Given the pivotal role that Th17 cells play
in mucosal homeostasis and epithelial barrier function (19), the
infection and depletion of a relatively small number of Th17 cells
could have a significant impact on mucosal immunity and the
clinical outcome of HIV infection. Additional in vivo work is
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FIGURE 7. CD11c+ DCs are required for E. coli-induced expansion and enhancement of infection of LP T cells. LPMCs were enriched for CD3+ T cells
by depletion of CD11c (DCs) and CD19 (B cells) LP cells (CD11c/CD19-depleted LPMC). CFSE-labeled LPMCs were cultured for 5 d with or without
heat-killed E. coli (5 E. coli:1 LPMC). (A) E. coli-induced proliferation of CD3+ CD4+ T cells was evaluated by assessing the percentage of CFSElo cells
within CD3+ CD4+ T cells in cultures stimulated with E. coli and are shown with background CD3+ CD4+ T cell proliferation detected within unstimulated
LPMC cultures removed (net; n = 1 colon, 3 jejunums). (B) Culture supernatants were collected and assayed for IFN-g and IL-17, with values expressed as
the net picogram per milliliter produced in response to E. coli stimulation after removing IFN-g or IL-17 amounts detected in unstimulated cultures. Each
symbol represents one sample (n = 1 colon, n = 3 jejunums). Statistical significance for (A) and (B) was determined using the paired t test. (C) Levels of
HIV-1 p24 detected within culture supernatants from total LPMCs, CD11c/CD19-depleted LPMCs, or CD11c-depleted LPMCs pre-exposed to CCR5tropic HIV-1Bal and cultured with or without HK E. coli (5 E.coli:1 LPMC) for an additional 3 d. Each individual symbol represents one sample (n = 3
jejunums). Statistical significance was determined using the repeated measures ANOVA (overall p value) with a Bonferroni multiple comparison test. *p ,
0.05. (D) Levels of HIV-1 p24 detected within culture supernatants from total LPMCs pre-exposed to CCR5-tropic HIV-1Bal and cultured with or without
E. coli (5 E.coli:1 LPMC) in the presence of purified mouse IgG2a (isotype) or purified anti–HLA-DR for an additional 3 d. Each individual symbol
represents one sample (n = 4 jejunums). Statistical significance was determined using the paired t test.
The Journal of Immunology
Acknowledgments
We thank the surgical teams of the Department of Surgery, University of
Colorado Hospital, for assistance in obtaining tissue samples and Dave Shugarts (AIDS Clinical Trials Group, Clinical Trials Unit, University of Colorado Anschutz Medical Campus) and Nancy Madinger and the staff of the
Clinical Microbiology Laboratory, University of Colorado Anschutz Medical Campus, for technical assistance.
Disclosures
The authors have no financial conflicts of interest.
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necessary to determine whether commensal bacteria enhance the
targeting of Th17 subsets in the setting of HIV-1 infection.
Although depletion of LP CD4 T cells during HIV infection is
likely multifactorial, our study provides one potential mechanism
by which translocation of selected enteric bacteria from the intestinal lumen into the LP could enhance the infection and depletion of LP CD4 T cells, especially those producing IL-17 or
coproducing IL-17 and IFN-g. On the basis of our results, we
propose that during early HIV infection—as HIV replicates in the
intestinal LP, resulting in inflammation and damage to the integrity of the epithelial barrier—increased exposure of LP CD4
T cells to translocating commensal bacteria would lead to the
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harder to capture in “snapshots” using clinical samples. However,
clinical studies are necessary to confirm that these in vitro findings
are relevant to mucosal pathogenesis in either early or chronic
HIV-1 infection. An understanding of the role of microbial
translocation in mucosal pathogenesis and its attendant mechanisms will be important in facilitating the development of therapeutic approaches aimed at blocking this process at the mucosal
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38.
COMMENSAL E. coli ENHANCES HIV INFECTION OF GUT Th17 CELLS