A preponderance of CCR5(+) CXCR4(+) mononuclear cells enhances gastrointestinal mucosal susceptibility to human immunodeficiency virus type 1 infection

Abstract

The gastrointestinal mucosa harbors the majority of the body's CD4(+) cells and appears to be uniquely susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We undertook this study to examine the role of differences in chemokine receptor expression on infection of mucosal mononuclear cells (MMCs) and peripheral blood mononuclear cells (PBMCs) by R5- and X4-tropic HIV-1. We performed in vitro infections of MMCs and PBMCs with R5- and X4-tropic HIV-1, engineered to express murine CD24 on the infected cell's surface, allowing for quantification of HIV-infected cells and their phenotypic characterization. A greater percentage of MMCs than PBMCs are infected by both R5- and X4-tropic HIV-1. Significant differences exist in terms of chemokine receptor expression in the blood and gastrointestinal mucosa; mucosal cells are predominantly CCR5(+) CXCR4(+), while these cells make up less than 20% of the peripheral blood cells. It is this cell population that is most susceptible to infection with both R5- and X4-tropic HIV-1 in both compartments. Regardless of whether viral isolates were derived from the blood or mucosa of HIV-1-infected patients, HIV-1 p24 production was greater in MMCs than in PBMCs. Further, the chemokine receptor tropism of these patient-derived viral isolates did not differ between compartments. We conclude that, based on these findings, the gastrointestinal mucosa represents a favored target for HIV-1, in part due to its large population of CXCR4(+) CCR5(+) target cells and not to differences in the virus that it contains.

FIG. 1

Infection of PBMCs and minced biopsies with CCR5-tropic HIV_SX and X4-tropic HIV_NL4-3 reporter viruses expressing mCD24. A total of 10⁶ PBMCs, prepared by Ficoll-Hypaque density gradient centrifugation, were infected at an MOI of 0.5. In parallel, four biopsies obtained endoscopically from the rectosigmoid region of the same patient were minced and infected with an equivalent amount of virus for 4 h, washed, and cultured for 5 days in RPMI supplemented with 10 IU of IL-2/ml. Isolated MMCs and PBMCs were analyzed by flow cytometry. (A) Using side scatter and CD3 fluorescence, T lymphocytes were analyzed after gating on forward and side scatter. HIV-1-infected CD3⁺ cells were identified by positive cell surface staining by anti-mouse CD24. A higher percentage of MMCs than PBMCs were infected with HIV-1, as shown by the greater number of CD24⁺ (HIV-1-infected) cells extending rightward. (B and C) HIV_SX and HIV_NL4-3 are the infected cell types (MMCs and PBMCs) on the x axis, and the percent CD3⁺ mCD24⁺ (HIV-1-infected) cells are on the y axis. A line is used to connect the percentage of infected PBMCs and MMCs from the same subject.

FIG. 2

CXCR4 and CCR5 expression on isolated PBMCs and MMCs. PBMCs prepared by Ficoll-Hypaque gradient and MMCs isolated from minced mucosal biopsies endoscopically obtained from the rectosigmoid region were studied flow cytometrically. CD45⁺ lymphocytes were analyzed after gating on forward and side scatter by staining for CXCR4 and CCR5. (A) Representative flow cytometry plots outlining chemokine receptor expression on PBMCs and MMCs. (B) Data compiled after analysis of 13 paired samples. The x axis outlines the chemokine receptor phenotypes (CXCR4⁺ CCR5⁻, CXCR4⁺ CCR5⁺, and CXCR4⁻ CCR5⁺), while the y axis specifies the percentage of the lymphocyte population with that phenotype. Error bars represent the means ± the standard error of the mean.

FIG. 3

Chemokine receptor expression on HIV-1-infected cells. A total of 10⁶ PBMCs, prepared by Ficoll-Hypaque density gradient centrifugation, were infected at an MOI of 0.5. In parallel, four biopsies obtained endoscopically from the rectosigmoid region were minced and infected with an equivalent amount of virus for 4 h, washed, and cultured for 5 days in RPMI supplemented with 10 IU of IL-2/ml. Isolated MMCs and PBMCs were analyzed by flow cytometry. T lymphocytes were analyzed, by using side scatter and CD3 fluorescence, after gating on forward and side scatter. HIV-1-infected CD3⁺ cells were identified by positive cell surface staining by anti-mouse CD24. The CD24-stained cells were then examined for expression of CCR5 and CXCR4. Graphs outlining the chemokine receptor expression on HIV-1-infected PBMCs and MMCs are shown for HIV_SX (A) and HIV_NL4-3 (B). The x axis outlines the chemokine receptor phenotypes (CXCR4⁺ CCR5⁻, CXCR4⁺ CCR5⁺, and CXCR4⁻, CCR5⁺), while the y axis shows the percentage of infected CD3 lymphocytes with that phenotype. Error bars represent the means ± the standard error of the mean.

FIG. 4

Comparison of replication of primary mucosa- and blood-derived viral isolates in MMCs and PBMCs. Primary HIV-1 isolates were cultured from PBMCs and four mucosal biopsies of HIV-1-infected subjects in the presence of PBMCs from HIV-1-seronegative donors stimulated by 5 μg of PHA/ml. HIV-1 replication was assessed every 3 days. Virus-containing supernatants (patient 1, 97 ng of p24; patient 2, 25 ng of p24; patient 3, 75 ng of p24) were used to infect MMCs and PBMCs of HIV-1-seronegative patients. Supernatant from these cultures was analyzed after 3 and 7 days for HIV-1 p24 protein by ELISA. The results are expressed in terms of nanograms of p24 protein detected per milliliter. The results from infections using three pairs of mucosa (♦)- and PBMC (▵)-derived viral isolates are shown. The x axis outlines the day 3 and day 7 results of infections of MMCs and PBMCs, while the y axis shows the amount of p24 detected in the culture supernatant.

FIG. 5

Comparison of chemokine receptor usage by primary mucosa- and blood-derived viral isolates. Primary HIV-1 isolates were cultured from the peripheral venous blood and four mucosal biopsies of HIV-1-infected subjects in the presence of PBMCs from HIV-1-seronegative donors stimulated by 5 μg of PHA/ml. HIV-1 replication was assessed every 3 days. Virus-containing supernatants from the patients' mucosa and PBMCs were used to infect human osteosarcoma cells stably transfected with human CD4 alone or with either CCR5 or CXCR4. GFP was cotransfected into these cells under the control of the HIV-1 long terminal repeat promoter. The viral tropism was determined by analyzing the infected cell lines flow cytometrically for expression of GFP, a finding indicative of successful HIV-1 entry and replication. The flow cytometric plots derived from infections of CXCR4- and CCR5-expressing osteosarcoma cell lines using mucosa- and PBMC-derived primary HIV-1 isolates are shown. The plots show GFP expression on the x axis, while the y axis represents FL-2. GFP-positive cells are delineated by the R2 gate formed after the analysis of each viral isolate with a parental osteosarcoma cell line that was not transfected with a chemokine receptor.