Human Immunodeficiency Virus (HIV) and Human Cytomegalovirus (HCMV) Coinfection of Infant Tonsil Epithelium May Synergistically Promote both HIV-1 and HCMV Spread and Infection

Abstract

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) and human cytomegalovirus (HCMV) may occur during pregnancy, labor, or breastfeeding. These viruses from amniotic fluid, cervicovaginal secretions, and breast milk may simultaneously interact with oropharyngeal and tonsil epithelia; however, the molecular mechanism of HIV-1 and HCMV cotransmission through the oral mucosa and its role in MTCT are poorly understood. To study the molecular mechanism of HIV-1 and HCMV MTCT via oral epithelium, we established polarized infant tonsil epithelial cells and polarized-oriented ex vivo tonsil tissue explants. Using these models, we showed that cell-free HIV-1 and its proteins gp120 and tat induce the disruption of tonsil epithelial tight junctions and increase paracellular permeability, which facilitates HCMV spread within the tonsil mucosa. Inhibition of HIV-1 gp120-induced upregulation of mitogen-activated protein kinase (MAPK) and NF-κB signaling in tonsil epithelial cells, reduces HCMV infection, indicating that HIV-1-activated MAPK and NF-κB signaling may play a critical role in HCMV infection of tonsil epithelium. HCMV infection of tonsil epithelial cells also leads to the disruption of tight junctions and increases paracellular permeability, facilitating HIV-1 paracellular spread into tonsil mucosa. HCMV-promoted paracellular spread of HIV-1 increases its accessibility to tonsil CD4 T lymphocytes, macrophages, and dendritic cells. HIV-1-enhanced HCMV paracellular spread and infection of epithelial cells subsequently leads to the spread of HCMV to tonsil macrophages and dendritic cells. Our findings revealed that HIV-1- and HCMV-induced disruption of infant tonsil epithelial tight junctions promotes MTCT of these viruses through tonsil mucosal epithelium, and therapeutic intervention for both HIV-1 and HCMV infection may substantially reduce their MTCT. IMPORTANCE Most HIV-1 and HCMV MTCT occurs in infancy, and the cotransmission of these viruses may occur via infant oropharyngeal and tonsil epithelia, which are the first biological barriers for viral pathogens. We have shown that HIV-1 and HCMV disrupt epithelial junctions, reducing the barrier functions of epithelia and thus allowing paracellular penetration of both viruses via mucosal epithelia. Subsequently, HCMV infects epithelial cells, macrophages, and dendritic cells, and HIV-1 infects CD4⁺ lymphocytes, macrophages, and dendritic cells. Infection of these cells in HCMV- and HIV-1-coinfected tonsil tissues is much higher than that by HCMV or HIV-1 infection alone, promoting their MTCT at its initial stages via infant oropharyngeal and tonsil epithelia.

Keywords: HIV; breast milk; human cytomegalovirus; mother to child transmission; tonsil mucosal epithelium.

FIG 1

HIV-1 tat- and gp120-induced disruption of tight junctions facilitates HCMV paracellular spread via polarized tonsil epithetical cells. (A) Polarized tonsil epithelial cells were treated with recombinant HIV-1_BAL tat and gp120 proteins (10 ng/ml of each). Untreated cells or inactive tat- and gp120-treated cells served as a control. Culture medium was changed every day to add fresh proteins, and TER was measured. (B) After 5 days of protein treatment, the paracellular permeability of polarized cells was measured by IgG leakage assay. (C) After 5 days of protein treatment, one set of cells was fixed and immunostained for the tight junction proteins claudin-1, occludin, and ZO-1 (all red). Cell nuclei were stained with Sytox green nucleic acid stain (green). Cells were analyzed using fluorescence microscopy (Nikon Eclipse E400). Magnification, ×630. (D and E) After 5 days of protein treatment, HCMV AD169 at an MOI of 1 was added to the apical surface of polarized cells. After 2, 4, or 6 h, basolateral medium was collected and used to infect HFF; 48 h later, cells were immunostained with HCMV gB antibody (D). One set of HFF was directly infected with input virus. HCMV gB-positive cells were counted and presented as a percentage of infection (E). (F and G) After 5 days of HIV-1 gp120 protein treatment or without treatment (control), tonsil epithelial cells were measured for TER (G, lower chamber) and then incubated with clinical strain HCMV VR1814 at an MOI of 1 from apical membranes of polarized cells. After 4 h, basolateral medium was collected and used to infect HFF. HCMV VR1814 infection of HFF was examined by immunostaining of HCMV IE1/2 expression (green) (F). Cell nuclei were stained with propidium iodide solution (red). Merged images are presented, and the yellow signal shows colocalization of IE1/2 with cell nuclei, which indicates HCMV infection. Infected cells were quantitatively evaluated and presented as a percentage of infection (G, top). (A, B, E, and G) Data are means and SD (n = 3) and represent three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (in comparison with the control groups). #, not detected.

FIG 2

Cell-free HIV-1 virions induce tight junction disruption of tonsil epithelial cells and increase HCMV paracellular spread. (A) Polarized tonsil epithelial cells were exposed to dually tropic HIV-1_SF33 for 5 days. Each day, the culture medium was changed to add fresh virus. Untreated cells served as a control. TER was measured every day. (B) After 5 days, one set of HIV-1_SF33-treated or untreated cells were fixed and immunostained for tight junction proteins claudin-1, occludin, and ZO-1 (all in red). Cell nuclei were stained with propidium iodide solution (red). Cells were analyzed using fluorescence microscopy. Magnification, ×400. (C and D) HCMV AD169 at an MOI of 1 was added to the apical surface (upper chamber) of HIV-1_SF33-treated or untreated polarized tonsil cells for 5 days. After 1, 2, 4, or 6 h of HCMV AD169 inoculation, the culture medium from the basolateral chamber was collected and used to infect HFF. HCMV AD169 infection in HFF was examined by immunostaining of HCMV gB (red). Cell nuclei were stained with Sytox green nucleic acid stains (green). Cells were analyzed using fluorescence microscopy (C). Magnification, ×400. HCMV gB-positive HFF were counted in 10 random microscopic fields, and the percentage of cells positive for gB was determined (D). (E, F, and G) Polarized tonsil epithelial cells from two independent donors were exposed to dually tropic HIV-1_SF33, R5-tropic HIV-1_SF170, or X4-tropic HIV-1_92UG029 for 5 days, and TER was measured (F and G, bottom). Cells were then incubated for 4 h with HCMV VR1814 at an MOI of 1 from the apical surface. Culture medium from the basolateral chamber was collected and used to infect HFF. HCMV VR1814 infection of HFF was detected by HCMV IE1/2 immunostaining (green) (E). Cell nuclei were stained with propidium iodide solution (red). Cells were analyzed by fluorescence microscopy. Magnification, ×600. Merged panes show the yellow signal that indicates colocalization of IE1/2 with cell nuclei, indicating HCMV infection. Infected cells were quantitatively evaluated and presented as a percentage of infection (F and G, top). (A, D, F, and G) Data are means and SD of triplicate values. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared with the control cells). #, not detected.

FIG 3

HIV-1-induced disruption of tonsil epithelial tight junctions increases HCMV infection. (A and B) Polarized tonsil epithelial cells were exposed for 5 days to cell-free HIV-1_SF33 and its active and inactive proteins tat and gp120. Untreated cells served as a control. Cells were then infected with HCMV VR1814 at an MOI of 1. After 3 days, one set of cells was coimmunostained for HCMV gB (red) and tight junction protein occludin (green) (A). Cell nuclei were counterstained with DAPI (blue). Cells were analyzed by fluorescence microscopy. Magnification, ×630. The next set of cells was lysed, and HCMV VR1814 infection was examined by Western blotting, which detected HCMV gB (B). Sample loading for the Western blot was controlled by detecting β-actin. Immunoblots were performed at least twice, and representative results are shown. (C) Polarized tonsil epithelial cells were exposed for 5 days to cell-free HIV-1_SF33 and its active and inactive proteins tat and gp120, as described for Fig. 3A. TER of polarized cells was then measured (top), and cells were infected with HCMV VR1814 at an MOI of 1. After 3 days, cells were immunostained for HCMV gB, and infected cells were quantitatively analyzed (middle). Culture medium containing HCMV VR1814 released from apical and basolateral membranes was collected, combined, and used to infect HFF (bottom). (D) Polarized tonsil epithelial cells were exposed for 5 days to dually tropic HIV-1SF33, R5-tropic HIV-1SF170, or X4-tropic HIV-192UG029 viruses and at day 5 the TER was measured (top). Cells were then infected with HCMV VR1814 at an MOI of 1 for 3 days. Cells were immunostained for HCMV gB, and gB-expressing cells were quantitatively evaluated (bottom). (E) Polarized tonsil epithelial cells were infected with HCMV VR1814 with or without HIV-1SF33. The next set of cells were treated with gp120 alone or gp120 with HCMV VR1814. After 5 days, the TER was measured. (C, D, and E) Data are means and SD from one of two or three independent experiments, each in triplicate (n = 3). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared with control cells). #, not detected.

FIG 4

HIV-1 gp120-induced disruption of tight junctions in tonsil epithelial cells may play a critical role in increasing HCMV infection through activation of MAPK and NF-κB signaling. (A) Polarized tonsil epithelial cells were exposed to cell-free HIV-1_SF33 and UV-inactivated HIV-1_SF33. In parallel experiments, cells were treated with recombinant gp120 and heat-inactivated gp120. After 5 days, TER was measured (bottom), and cells were then infected with HCMV VR1814 at an MOI of 1 for 3 days. Cells were immunostained for HCMV gB and quantitatively evaluated (top). (B) Polarized tonsil epithelial cells were treated for 5 days with recombinant gp120 proteins from HIV-1_BAL, HIV-1_IIIB, HIV-1_CN54, and HIV-1_96ZM65. Untreated cells served as a control. After 5 days, TER was measured (bottom). Cells were then infected with HCMV VR1814 at an MOI of 1, and 3 days later, infection was examined by gB immunostaining, and gB-positive cells were quantitatively analyzed and presented as a percentage of infected cells (top). (C) Tonsil polarized epithelial cells from 10 independent donors were treated with HIV-1_BAL gp120 for 5 days, and TER was measured. Cells were then infected with HCMV VR1814 at an MOI of 1, and infection was quantified by counting gB-expressing cells (top). (D and E) Polarized tonsil epithelial cells were treated with gp120 and tat proteins in the presence or absence of the chemical compounds U0126 and MG-132, which are inhibitors of MAPK and NF-κB, respectively. After 5 days, TER was measured (bottom), and cells were then infected with HCMV VR1814 at an MOI of 1. After 3 days, one set of cells was quantitatively evaluated for HCMV infection by gB immunostaining (top) (D). The next set of cells was analyzed for expression of HCMV IE1/2, and total and phosphorylated ERK1/2, IκB, and NF-κB were measured by Western blotting assay (E). (F) Polarized tonsil epithelial cells were treated with HIV-1 gp120 for 4 days and then maintained without gp120 for the next 4 days. Control cells were not treated with gp120. TER was measured every day (left). One set of cells treated with gp120 for 4 days and one set of cells untreated for the next 4 days were infected with HCMV VR1814. Untreated control cells were also infected with HCMV VR1814 at days 4 and 8. HCMV VR1814-infected cells were immunostained for gB, and gB-positive cells were counted (right). (A, B, C, D, and F) Data represent one of three independent experiments and are means and SD of triplicate values. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared with control cells).

FIG 5

HCMV infection of ex vivo infant tonsil explants. (A) Explants were placed in Transwell inserts with the mucosal surface facing up to be accessible for viral inoculation, and the lateral edges were sealed with 4% agarose. Tissues were maintained for 6 days, and culture medium was changed every 2 or 3 days. At the end of each day, one set of explants was fixed, sectioned, and immunostained for the tight junction protein occludin (red). Cell nuclei were stained for DAPI (blue). Cells were analyzed by fluorescence microscopy. Magnification, ×400. EP, epithelium; LP, lamina propria. (B) Polarized-oriented infant tonsil explants were infected with 3 × 10⁵ IU of HCMV VR1814 from the mucosal (apical) surface of tonsil epithelium. After 1 day, one set of explants was immunostained for HCMV IE1/2, and after 3 and 5 days, one set of explants was immunostained for HCMV gB (both green). Cell nuclei were counterstained with DAPI (blue). Cells were analyzed by fluorescence microscopy. Magnification, ×100 (two lower left panels) and ×400 (all others). (C and D) Tonsil explants from three independent donors were infected with HCMV VR1814. Uninfected explants served as a control. After 3, 6, and 9 days, culture medium was collected from the upper and lower chambers, combined, and used to infect HFF with HCMV. Infection was quantified by IE immunostaining (C). Data are means and SD (n = 3). At day 9, the infected and control tissue explants were homogenized, lysed, and examined for HCMV gB and IE1/2 by Western blotting (D).

FIG 6

HIV-1 tat and gp120 disrupt tonsil epithelial junctions and facilitate HCMV infection. (A) Tonsil tissue explants were treated with active or inactive HIV-1 tat and gp120 proteins for 5 days; culture medium was changed daily to add fresh proteins. After each day, one set of explants was immunostained for occludin (red). (B) One set of tonsil explants treated with active tat and gp120 for 3 days was infected with 3 × 10⁵ IU of HCMV VR1814; after 2 days, tissues were coimmunostained for HCMV gB (green) and occludin (red). Cell nuclei were stained with DAPI (blue), and cells were analyzed by fluorescence microscopy. Magnification, ×400. EP, epithelium; LP, lamina propria. (C) Tonsil explants from two independent donors were treated with active tat and gp120. After 3 days, explants were infected with 3 × 10⁵ IU of HCMV VR1814. After 2 more days, tissues were immunostained for HCMV gB; gB-expressing cells were counted, and values are presented as a percentage of infection. (D) Tonsil tissues from six donors (#1 to #6) were treated with HIV-1 active or inactive tat and gp120 for 3 days. Untreated tissues served as a control. Tissues were then infected with HCMV VR1814 for 3 days. Tissues were immunostained for HCMV gB, and HCMV gB-expressing cells were quantitatively evaluated. (A and B) Data are means and SD of triplicate values. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared with the controls treated with inactive tat+gp120).

FIG 7

HCMV infection disrupts tonsil epithelial junctions and facilitates paracellular HIV-1 spread. (A and B) Polarized tonsil epithelial cells were infected with 1, 2, or 3 MOI of HCMV VR1814, and TER was measured after 3, 4 and 5 days (A). One set of cells infected with HCMV VR1814 at an MOI of 3 was coimmunostained for occludin (red) and HCMV gB (green) after 3, 4, or 5 days (B). Uninfected polarized cells served as a control. Cell nuclei were stained with DAPI (blue), and cells were analyzed by fluorescence microscopy. Magnification, ×400. (C) Polarized tonsil epithelial cells from two independent donors were infected with HCMV VR1814 for 5 days. TER was measured (bottom), and the apical surface of cells was exposed for 2 h to dually tropic HIV-1_SF33, R5-tropic HIV-1_SF170, and X4-tropic HIV-1_92UG029 (3 ng/insert). Culture medium from the basolateral surface was collected and examined for p24 (top) using ELISA. Culture medium was also tested for HIV-1 infectivity in PBMC (middle). (A and C) Data are means and SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared with the control cells). #, not detected.

FIG 8

HCMV and HIV-1 coinfection of ex vivo tonsil tissues. (A) Polarized-oriented tonsil explants were infected with HCMV VR1814 or HIV-1_SF33 or coinfected with both viruses. Uninfected tissues served as a control. After 5 days, tissue sections were coimmunostained for HCMV gB (green) and HIV-1 p24 (red). (B) HCMV VR1814 and HIV-1_SF33 -coinfected tissues were costained for HCMV gB (green) and for markers of macrophages CD68 and DC CD1c (both red). (C) HCMV VR1814- and HIV-1_SF33-coinfected tissues were costained for HIV-1 p24 (green) and for markers of CD4 T lymphocytes (CD4), macrophages (CD68), and DC (CD1c) (all red). Merged panels are shown, and the yellow signal indicates HCMV or HIV infection of CD4 T lymphocytes, macrophages, and DC. Cell nuclei were stained with DAPI (blue), and cells were analyzed by confocal microscopy. Magnification, ×400. EP, epithelium; LP, lamina propria. (D and E) Tonsil explants from two independent donors were infected with HCMV VR1814 or HIV-1_SF33 or coinfected with both. After 5 days, tissue sections were costained for HCMV gB (green) and for markers of macrophages and DC (red). In parallel experiments, tissue sections were immunostained for HIV-1 p24 (green) and for markers of CD4 T lymphocytes, macrophages, and DC (red). HCMV-infected macrophages and DC (D), and HIV-1-infected CD4 T lymphocytes, macrophages, and DC (E) were quantitatively evaluated and are presented as number of cells per mm². Similarly, HCMV-infected epithelial cells also were quantitatively evaluated. Data are means and SD. *, P < 0.05; **, P < 0.01 and ***, P < 0.001; ****, P < 0.0001 (explants coinfected with both viruses were compared with explants infected with only HIV-1 or HCMV).

FIG 9

HCMV and HIV-1 coinfection of tonsil tissues synergistically promotes infection of both viruses. (A) Tonsil tissue explants from four independent donors were infected with HIV-1_SF33, and tissues were cultured with or without GCV or lamivudine for 9 days. The next set of tissue explants from the same donors were coinfected with HIV-1_SF33 and HCMV VR1814 in the presence or absence of GCV. After 3, 6, and 9 days, culture medium was collected and examined for HIV-1 p24 using ELISA. p24 values were normalized per 50 mg of tissue. (B) Tonsil explants from three independent donors were infected with HCMV VR1814, and tissues were cultured with or without GCV or lamivudine for 9 days. The next set of tonsil explants was coinfected with HCMV VR1814 and HIV-1_SF33 with or without GCV. Tissue culture medium was collected after 3, 6, and 9 days and tested for HCMV infection of HFF. HCMV IE-positive HFF were quantitatively evaluated and are presented as a percentage of HCMV-infected cells (normalized per 50 mg of tissue). (C) Tonsil tissue explants from two independent donors were infected with only HCMV VR1814 or coinfected with HCMV VR1814 and dually tropic HIV-1_SF33, R5-tropic HIV-1_SF170, or X4-tropic HIV-1_92UG029 at 3 ng/tissue. At 3, 6, and 9 days of culture, the medium was collected and tested for HIV-1 p24 by ELISA. All data are means and SD of triplicate values. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared with coinfected explants and explants infected with only HIV-1 or HCMV). #, not detected.

FIG 10

Model showing how HIV-1 and HCMV coinfection of tonsil tissues may synergistically enhance both viral infections. (A) Well-developed tight and adherens junctions of oropharyngeal and tonsil epithelia play a critical role in the establishment of barrier function, which prevents the paracellular spread of viral pathogens, including HIV-1 and HCMV. (B) Most MTCT of HIV-1 and HCMV may occur during labor and breastfeeding, when these viruses may simultaneously interact with mucosal epithelial cells, leading to disruption of their integrity. Cell-free HIV-1 from cervicovaginal secretions and breast milk may induce the disruption of oropharyngeal and tonsil mucosal epithelium junctions by gp120 on the viral surface. Furthermore, in HIV-1-infected infants, the secreted tat may also disrupt mucosal tight junctions. Thus, HIV-1-induced disruption of oropharyngeal mucosal epithelia may enhance HCMV paracellular spread, initiating HCMV MTCT. HCMV infection of oropharyngeal mucosal epithelium also disrupts oropharyngeal and tonsil mucosal epithelia and enhances HIV-1 paracellular spread, which may initiate HIV-1 MTCT. (C) HIV-1- and HCMV-induced disruption of mucosal epithelium integrity may promote the paracellular spread of HIV-1, increasing its accessibility to intramucosal and submucosal CD4 T lymphocytes, macrophages, and DC. HIV-1- and HCMV-induced disruption of mucosal epithelium also enhances HCMV paracellular spread and infection of epithelial cells, which subsequently spread HCMV to intraepithelial and submucosal macrophages and DC. Thus, HIV-1 and HCMV viruses may synergistically promote MTCT through infant tonsil mucosal epithelium.