Microbial translocation from the gastrointestinal tract has been proposed as a causal link to chronic immune activation during progressive HIV-1 infection.1 Recent data have implicated the loss of immunological defenses2-4 and the physical breakdown of the intestinal barrier5-7 as mechanisms that enhance translocation of bacteria and their products into the gut lamina propria. Here we provide evidence of a new mechanism in HIV-1 infection in which a cell surface protein known as syndecan-1 (SYN1) facilitates microbial translocation across an intestinal epithelial barrier compromised via the aberrant expression of the pore-forming, tight junction (TJ) protein, claudin-2 (CLD2).
SYN1 is one of 4 members of a family of cell surface heparin-sulfate proteoglycans expressed with temporal and spatial specificities on various cell types within many different tissues and involved in a wide array of cellular processes.8 SYN1 is primarily expressed on the basolateral surface of intestinal epithelium9 and also on plasma cells10 and plays a role in cell-cell and cell-matrix interactions, migration, proliferation, and cell differentiation.8 Increasing in vitro evidence has linked SYN1 with gut microbial translocation through the ability of bacteria to bind SYN1's heparin sulfate (HS) motifs.11-15 However, the ability of luminal bacteria to interact with SYN1 is likely limited in healthy individuals due to its basolateral location but can take on increasing importance in persons with increased intestinal permeability, such as those individuals infected with HIV-1.5-7
Thus, when we identified SYN1 from a recent microarray analysis as a gene increased in expression during chronic HIV-1 infection,16 we investigated a potential role for SYN1 in microbial translocation during HIV-1 infection. In this report, we indeed show that there is both an increase in intestinal permeability during HIV-1 infection through the upregulation of CLD2 and a strong association between SYN1 expression in the intestinal epithelium and microbial translocation and systemic immune activation.
Gut and Lymph Node Tissue From HIV-1-Infected Individuals
Ileal, rectal, and inguinal lymph node biopsies from 18 HIV-1-infected subjects at different clinical stages and 4 uninfected subjects were obtained for this University of Minnesota institutional review board-approved study. Tissues were fixed in paraformaldehyde or Streck's Tissue Fixative before embedding in paraffin.
Microarray Analysis of Inguinal Lymph Nodes
RNA extractions, synthesis of biotin-labeled cRNA probes, and microarray hybridization followed previously published procedures.16
Immunohistochemistry and immunofluorescence were performed as previously described17 using a biotin-free detection system on 5 μm tissue sections mounted on glass slides. Tissues were deparaffinized and rehydrated in deionized water. Heat-induced epitope retrieval was performed using either a water bath (95-98°C for 10-20 minutes) or high pressure cooker (120°C for 30 seconds) in one of the following buffers: Diva Decloaker (Biocare Medical, Concord, CA), 10 mM sodium citrate, pH 6.0, or 1 mM EDTA, pH 8.0, followed by cooling to room temperature. Tissues sections were blocked with SNIPER Blocking Reagent (Biocare Medical) for 15 minutes at room temperature. Endogenous peroxidase was blocked with 3% (v/v) H2O2 in methanol. Primary antibodies were diluted in Tris-NaCl-blocking buffer (0.1M Tris-HCl, pH 7.5; 0.15M NaCl; 0.05% Tween 20 with Dupont blocking buffer) and incubated overnight at 4°C. After the primary antibody incubation, sections were washed with phosphate-buffered saline. Sections for immunofluorescence were then incubated with rabbit or mouse fluorophore-cojugated secondary antibodies in 5% nonfat milk for 2 hours at room temperature. These sections were washed, nuclei counterstained blue with TOTO-3, and mounted using Aqua Poly/Mount (Polysciences, Inc, Warrington, PA). Sections for immunohostochemistry were incubated with mouse or rabbit polymer system reagents conjugated with horseradish peroxidase (Dako Cytomation, Carpinteria, CA) according to the manufacturer's instructions, developed with 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA), counterstained with Harris hematoxylin (Surgipath Medical, Richmond, IL), and mounted using Permount (Fisher Scientific, Pittsburgh, PA). Stained sections were examined either by light microscopy or immunofluorescent confocal microscopy at ambient temperatures. Light micrographs were taken using an Olympus BX60 upright microscope with the following objectives: ×10 (0.3 NA), ×20 (0.5 NA), and ×40 (0.75 NA); images were acquired using a Spot color mosaic camera (model 11.2) and Spot acquisition software (version 4.5.9; Diagnostic Instruments). Immunofluorescent micrographs were taken using an Olympus BX61 Fluoview confocal microscope with the following objectives: ×20 (0.75 NA), ×40 (0.75 NA), and ×60 (1.42 NA); images were acquired using Olympus Fluoview software (version 1.7a).
To quantify levels of SYN1 expression in the gut, 16 randomly stained images from each specimen were captured and positive cells enumerated using Photoshop (CS2, version 9.0; Adobe Systems) with plug-ins from Reindeer Graphics. Specifically, this program utilizes a threshold tool to set a gray level that discriminates positively stained cells from background and marks the positive signal with a red overlay. The program then measures the area of the signal above the threshold, expressed as a % of the total area. Images for SYN1 quantification focused on the gut epithelium to minimize the positive signal derived from SYN1 expression on plasma cells residing within the lamina propria. Data were expressed as % tissue area positive for SYN1.
A semiquantitative approach was used to measure CLD2 expression. Stained images of the entire tissue section were captured from each specimen and CLD2 measured as a function of its expression in the luminal epithelial cells (the outer layer/perimeter of epithelium exposed to the intestinal lumen). The amount of CLD2 expression along the epithelial perimeter was determined and expressed as a range of % tissue area positive for CLD2 (eg, >90% indicates that CLD2 was expressed in more than 90% of the luminal epithelial cells for a particular gut section).
To quantify numbers of translocated E. coli in the inguinal lymph node, 16 randomly stained images from each specimen were captured and positive cells enumerated by manually counting positively stained bacteria in each image. The average number of bacteria per lymph node area was determined and log transformed. Data were expressed as log10E. coli/mm2 of tissue. Isotype-matched IgG negative control antibodies in all instances yielded negative staining results (see Table, Supplemental Digital Content 1,http://links.lww.com/QAI/A76 which lists the primary antibodies and antigen retrieval methodologies).
Fluorescence In-Situ Hybridization
Cy3-conjugated fluorescence in-situ hybridization (FISH) probes (EUB338: GCTGCCTCCCGTAGGAGT; NON338: ACTCCTACGGGAGGCAGC) were obtained from Eurofins MWG/Operon and used to detect bacterial 16s rRNA according to a procedure previously published.18 Briefly, tissues were deparaffinized and rehydrated to 70% ethanol. Tissues were then immersed in 0.5M EDTA for 15 minutes at 37°C; in 5 μg/mL proteinase K (Boehringer Mannheim) in 100 mM Tris-HCl plus 50 mM EDTA, pH 7.5, for 30 minutes at 37°C; and in 70% ethanol for 1 minute. After dehydration in ethanol, tissue sections were air dried, and a 13-μL probe mixture containing 1 μg/mL of the respective probe in hybridization mixture (0.45M NaCl; 45 mM sodium citrate, pH 7.0; 25% formamide; 10× Denhardt's solution; 10% dextran sulfate; 1% Triton X-100; 0.1% sodium dodecyl sulfate) was applied to the sections overnight at 40°C in a humidified chamber. Sections were washed first in 0.1M Tris-HCl plus 0.9M NaCl, pH 7.5 for 20 minutes at 48°C, followed by phosphate-buffered saline. Nuclei were counterstained blue with DAPI and mounted using Aqua Poly/Mount (Polysciences Inc.). Immunofluorescent micrographs were taken using an Olympus BX61 Fluoview confocal microscope.
To test for comparisons of SYN1 across stage, we used a mixed effects model that has random intercepts specific to each subject. Parameter estimates are obtained using restricted maximum likelihood, and all P values use a normal approximation to the sampling distribution of the Wald statistics specific for each model parameter. To test for an overall effect for stage, a contrast was formed that allowed for testing for a difference between all groups and uninfected subjects. The P value for this overall effect is 0.0003. To test for differences between the 3 infected stages and uninfected individually, we used the Wald test statistic specific to the parameter for that stage. We did not detect statistically significant differences between any of the 3 positive stages using this model. To test for CLD2 differences across stage, the Analysis of Variance method was used.
All associations were tested using a mixed effects model that has random intercepts specific to each subject to model correlation among measurements from the same subject. A normal approximation to the sampling distribution of the restricted maximum likelihood estimates was used to determine P values. A logarithmic transformation was employed for analysis of all continuous variables to reduce the effect of potential influential observations and reduce dependence of variances on mean levels. All computations were conducted using S-PLUS Version 3.4 distributed by MathSoft, Inc (Cambridge, MA).
Gut Expression of SYN1 is Increased During HIV-1 Infection
To establish a linkage between SYN1 and microbial translocation, we first determined the cellular/spatial localization and expression levels of SYN1 in the gut. We stained for SYN1 in ileal and rectal biopsies from uninfected and HIV-1-infected individuals in each clinical stage of disease-acute (defined as individuals infected within 4 months of documented seroconversion), presymptomatic (defined as individuals infected for at least 4 months with a CD4+ T-cell count >200 cells/μL), and AIDS (defined as infected individuals with a CD4+ T-cell count <200 cells/μL) (Table 1). SYN1 was present at basal levels in uninfected individuals (Figs. 1A, B) but steadily increased in the mucosal epithelium (Figs. 1C, D) of the large and small intestine during the progression of HIV-1 infection, with highest levels in the AIDS stage of disease (Figs. 1C, D). SYN1 was also present in leukocytes, primarily CD27+ plasma cells (see Figure, Supplemental Digital Content 2,http://links.lww.com/QAI/A77 which shows SYN1 and CD27 colocalization in leukocytes), residing within the lamina propria. The levels of SYN1 were quantified, revealing significant increases at all stages of HIV-1 infection: ∼1.6-fold increase in the acute stage of disease and a larger 2.7-3.4 fold increase (P ≤ 0.047) in the presymptomatic and AIDS stages of disease (Fig. 1E).
Increased CLD2 Expression Begins in Early HIV-1 Infection and is Coexpressed with SYN1 in the Intestinal Epithelium
The maintenance and preservation of an intact intestinal epithelial barrier is important in limiting microbial access to the basolateral surface of epithelial cells and the host's immune cells residing within the lamina propria. Key players in this process are the claudins, tight junction proteins involved in establishing the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells.19 Aberrant expression of TJ proteins and compromised barrier function have been reported for individuals in a number of different settings, such as inflammation19,20 and virus infections,21-25 including HIV-1.7 Because altered expression of claudins could potentially impair barrier protection and increase paracellular permeability, providing bacteria potential access to SYN1, a mechanism which could enhance microbial translocation, we investigated the expression of claudins (claudins-1, -2, and -4) in the gut of uninfected and HIV-1-infected individuals. We found a substantial upregulation of the pore-forming junctional protein, CLD2,26 along the extracellular surface of the intestinal epithelium and crypts (Fig. 2A) beginning in early infection (acute stage) and continuing throughout disease progression (Fig. 2B) although expression of claudins-1 and -4 were essentially unchanged (Fig. 2C). Moreover, CLD2 often was coexpressed with SYN1, CLD2 along the apical surface of the epithelium and SYN1 along the basolateral surface (Fig. 2D). Thus, increased expression of CLD2, a junctional protein shown to increase paracellular permeability26 and promote TJ strand discontinuities,27 during HIV-1 infection could compromise barrier protection, potentially allowing microbes access to the basolateral location of SYN1.
SYN1 Expression is Associated with Increases in Microbial Translocation and Systemic Dissemination
We tested the relationship between microbial translocation and SYN1 expression in vivo during HIV-1 infection by visualizing translocated bacteria (using an antibody recognizing E. coli) and SYN1-expressing cells in gut biopsies from HIV-1-infected persons. As shown in representative images in Fig. 3A, translocated E. coli often colocalized with SYN1-expressing epithelial cells in the gut of HIV-1-infected individuals. To confirm detection of translocated bacteria, we also employed fluorescence in situ hybridization (FISH) using a universal bacterial 16s ribosomal RNA probe and found general agreement between immunofluorescence and FISH detection methods (see Figure, Supplemental Digital Content 3,http://links.lww.com/QAI/A78 which shows FISH-detected bacteria in the lamina propria of the gut). Isotype control antibodies yielded negative staining results (see Figure, Supplemental Digital Content 4,http://links.lww.com/QAI/A79 which shows negative staining results when isotype-matched antibodies are used in tissue specimens).
Bacteria that translocate into the gut lamina propria can then spread systemically via the lymphatic system to distal organ sites. We found evidence that SYN1 expression not only was associated with enhanced microbial translocation in the gut but also with dissemination of bacteria. We discovered a significant positive correlation between gut expression of SYN1 and numbers of disseminated bacteria in inguinal lymph nodes (r = 0.6893, P < 0.0001) (Fig. 3B). As physical damage and breaches in the intestinal epithelium could be an alternative or additional mechanism of microbial translocation into the lamina propria, we used cytokeratin-specific antibodies to microscopically examine the structural integrity of the intestinal epithelium. We detected little morphological evidence of extensive epithelial cell damage or widespread breaches in the continuous layering of cytokeratin+ columnar epithelial cells (Fig. 3C). Moreover, in those areas of the gut with discrete focal breaks, we found little accumulation of bacteria in the immediate vicinity. We thus conclude from these data and previous in vitro findings11-15 that increased SYN1 and CLD2 expression in the intestinal epithelium during HIV-1 infection could be one mechanism facilitating gut microbial translocation and subsequent spread systemically.
SYN1 Expression is Associated With Both Systemic Immune Activation and Antibacterial Responses
The host responds to increases in bacteria and microbial immunogens such as lipopolysaccharide (LPS) and bacterial DNA through a number of regulatory mechanisms-IL-17 is an increasingly important antibacterial cytokine involved in inhibiting replication and dissemination of bacteria,28 whereas myeloid differentiation factor 2 (MD-2) is the essential sensor of LPS for Toll-like receptor 4 signaling, whose levels increase in response to bacterial infection.29 We found a significant positive correlation between SYN1 expression in the gut and mRNA levels of both IL-17 and MD-2 in the inguinal lymph node (r = 0.3709 and 0.3052, respectively, P ≤ 0.05) (Fig. 4A and B). Additionally, we found a relationship between numbers of disseminated bacteria and mRNA levels of both IL-17 and MD-2 in the inguinal lymph node (Figs. 4C, D); however, the latter analysis was limited and not powered to detect significance. These data indicate an antibacterial host response in the lymph nodes, providing supporting evidence of systemic microbial translocation via a SYN1-dependent pathway.
As previous studies have reported the ability of microbial products such as LPS and bacterial DNA to activate immune cells in vitro30 and in vivo,1,31-33 we wanted to determine whether there was a linkage between microbial translocation and systemic immune activation as measured in distal lymphoid tissue. First, we compared the extent of microbial translocation (numbers of translocated E. coli in lymph nodes) to the mRNA expression levels of the cell activation marker Ki-67 in inguinal lymph nodes and found a significant positive correlation between systemic microbial translocation and cell activation (r = 0.4061, P = 0.0034) (Fig. 5A). Second, we observed high levels of immune activation in lymph nodes in spatial conjunction with locally high numbers of translocated bacteria. In representative images in Figures 5B and C, there are numerous E. coli within an inguinal lymph node associated with increased numbers of Ki-67-expressing cells in an HIV-1-infected person compared with uninfected individuals (Fig. 5D), in which translocated bacteria were not detected. Third, we found a significant positive correlation between IFN-α expression and numbers of translocated bacteria in lymph nodes (r = 0.6709, P = 0.0071), immune stimulation consistent with Toll-like receptor recognition of microbial products34 (Fig. 5E). These data support a mechanism of widespread cell activation and antibacterial responses during HIV-1 infection in which SYN1 expression in the gut may be augmenting microbial translocation and systemic spread of bacteria.
Levels of SYN1 and Microbial Translocation are Inversely Correlated With CD4+ T-Cell Survival
Microbial translocation has been purported as a contributing factor to CD4+ T-cell loss during HIV-1 infection due to its role in chronic immune activation and reports showing an inverse correlation between the levels of microbial translocation and numbers of CD4+ T cells.1,33,35,36 We therefore wanted to determine whether SYN1 expression and augmented microbial translocation was linked to CD4+ T-cell numbers in the peripheral blood. We indeed found inverse correlations between SYN1 expression in the gut and peripheral blood CD4+ T cells (r = −0.3156, P = 0.001) (Fig. 6A) and between the extent of microbial translocation (numbers of translocated E. coli in lymph nodes) and peripheral blood CD4+ T cells (r = −0.4745, P = 0.001) (Fig. 6B), consistent with SYN1-induced microbial translocation contributing directly or indirectly to CD4+ T-cell depletion as measured in the peripheral blood.
To examine this in the contextual environment of lymphatic tissue, we used immunofluorescence to observe CD4+ T cells present in inguinal lymph nodes from HIV-1-infected individuals with divergent levels of microbial translocation (Fig. 6B). We found a similar relationship between the extent of microbial translocation and lymph node CD4+ T cells, whereby greater loss of CD4+ T cells was detected in lymph nodes coincident with higher levels of microbial translocation (compare Figs. 6C, D). Furthermore, we found an increase in CD4+ T-cell apoptosis in lymph nodes harboring greater numbers of translocated bacteria (compare Figs. 6C, D), consistent with previous reports showing LPS and other microbial products as inducers of activation-induced apoptosis in lymphocytes in vitro30 and in vivo.32,37 These data suggest that microbial translocation may be a contributing factor in chronic immune activation and CD4+ T-cell loss both in the periphery and lymphatic tissue during HIV-1 infection.
Increased levels of microbial translocation during HIV-1 infection have been well documented1,31,33,35,36,38-40 and ascribed to such causes as the loss of immunological defenses2-4 and the physical breakdown of the intestinal barrier.5-7 Here, we provide evidence of a new mechanism linking HIV-1 infection-associated increases in gut epithelial cell SYN1 and the pore-forming, TJ protein CLD2 to increased bacterial translocation in the gut and systemic spread into distal sites such as peripheral lymph nodes. This increase in microbial translocation is associated with antibacterial responses, measured as correlated increases in such mRNA markers (IL-17 and MD-2), which may explain why the presence of bacteria systemically is not associated with gram-negative sepsis.
The mechanism by which SYN1 increases bacterial translocation has been reported to involve interactions between bacterial surface proteins and HS glycosaminoglycan motifs surfacing the extracellular domain of SYN1-expressing cells.11-15 However, these HS motifs are likely shielded from luminal bacteria in healthy individuals due to the basolateral positioning of SYN1 along the intestinal epithelium. The dramatic increase in CLD2 during HIV-1 infection would provide an initial breach in barrier function, increasing permeability,26 and allowing potential microbial access to the HS motifs of SYN1, which then can facilitate microbial translocation across the epithelial barrier and into the lamina propria.
The polarized epithelium of the host plays a significant role in resistance to bacterial infection, whereby disruption/alteration of the claudin composition of TJ's can lead to imbalance of this equilibrium and increase paracellular permeability.41 Bacteria have been shown to take advantage of “leaky” TJ's, gaining access to the basolateral surfaces of the epithelium, ultimately increasing invasiveness and paracellular translocation.42-48 Moreover, CLD2 has been implicated as one of the primary claudin determinants in epithelial barrier dysfunction and increased paracellular permeability in vivo49-51 by decreasing the “tightness” of individual claudin-based TJ strands27,52; finally, the expression of CLD2 has also been associated in vivo with bacterial sepsis.53
Despite CLD2's role in increasing gut permeability and its initial upregulation in acute/early infection, we did not find a direct relationship between CLD2 expression levels and microbial translocation, consistent with previous observations in which microbial translocation is not appreciable, as detected experimentally, until chronic HIV-1 infection.1,31,36 The extent of microbial translocation is greatest in the presymptomatic/AIDS stages of disease when SYN1 levels are most upregulated (Fig. 3B). This then suggests that CLD2 may be required for SYN1 accessibility, but SYN1 is the agent that actively facilitates microbial translocation. Although this mechanism in the context of HIV-1 infection deserves further study, what is clear from our data is that SYN1-mediated microbial translocation differs markedly from other proposed mechanisms involving the loss of immunological defenses2-4 or physical breakdown of the intestinal barrier (increased apoptosis).5-7 We think that both the direct SYN1 pathway and these indirect mechanisms may work in concert to increase microbial translocation during HIV-1 infection.
We show that the extent of microbial translocation is associated with both systemic immune activation, measured as correlated increases in such mRNA markers (Ki-67 and IFN-α) and the close proximity of translocated E. coli to activated immune cells within inguinal lymph nodes, and apoptosis of CD4+ T cells in lymphatic tissue. Although it remains difficult to dissect pathogenic contributions from the virus itself vs. bacteria and their immunogenic components, these data, nevertheless, suggest that microbial translocation may be a contributing factor in chronic immune activation and CD4+ T-cell depletion.
Although our study and previous reports suggest that SYN1-expressing cells can augment microbial translocation by enhancing bacterial binding and subsequent internalization into intestinal epithelial cells,11-15 we suspect that this is an adverse off-target effect of SYN1's physiological functions in HIV-1-infected lymph nodes where increased expression was first detected.16 We further speculate, based on SYN1's role in cell-cell and cell-matrix interactions,8 that SYN1's increased expression in the intestinal epithelium and infiltration of SYN1+ plasma cells into the gut during HIV-1 infection may be related to inflammation and/or tissue remodeling.8,54,55 Because of the previously documented relationship between lymphatic tissue collagen deposition and CD4+ T-cell depletion,56 we will be particularly interested in future studies exploring the relationship of SYN1 to these immunopathological changes in which SYN1 might be both involved in the fibrotic changes and the chronic immune activation that drives them.
We thank all of the donor participants in this study.
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