Epithelium-innate immune cell axis in mucosal responses to SIV

Abstract

In the SIV (simian immunodeficiency virus)-rhesus macaque model of HIV-1 (human immunodeficiency virus type I) transmission to women, one hallmark of the mucosal response to exposure to high doses of SIV is CD4 T-cell recruitment that fuels local virus expansion in early infection. In this study, we systematically analyzed the cellular events and chemoattractant profiles in cervical tissues that precede CD4 T-cell recruitment. We show that vaginal exposure to the SIV inoculum rapidly induces chemokine expression in cervical epithelium including CCL3, CCL20, and CXCL8. The chemokine expression is associated with early recruitment of macrophages and plasmacytoid dendritic cells that are co-clustered underneath the cervical epithelium. Production of chemokines CCL3 and CXCL8 by these cells in turn generates a chemokine gradient that is spatially correlated with the recruitment of CD4 T cells. We further show that the protection of SIVmac239Δnef vaccination against vaginal challenge is correlated with the absence of this epithelium-innate immune cell-CD4 T-cell axis response in the cervical mucosa. Our results reveal a critical role for cervical epithelium in initiating early mucosal responses to vaginal infection, highlight an important role for macrophages in target cell recruitment, and provide further evidence of a paradoxical dampening effect of a protective vaccine on these early mucosal responses.

FIGURE 1

Co-clustering of CD4 T cells (white) and SIV vRNA⁺ infected cells (red) in cervical mucosa close to the endocervical epithelium after vaginal challenge. The montage was constructed with images (original magnification ×200) of the cervical tissue from a rhesus macaque 7 days after vaginal infection with SIVmac251. Insert shows co-clustering in the marked region at higher magnification. The vRNA+ cells are predominantly CD4+ T cells (co-localized red and white).

FIGURE 2

Kinetics of accumulation of CD4⁺ T cells and vRNA+ cells beneath the epithelium of cervical transformation zone and adjoining endocervix. (A) Cervical CD4 T cells remained at basal levels at 3 d.p.i. but their numbers were significantly increased by 7 d.p.i. and peaked approximately at 14 d.p.i. before declining to levels still considerably higher than baseline levels by 28 d.p.i. (B) Representative images from 3 different animals for each time point in the kinetics of recruitment of brown-stained CD4 T cells beneath the endocervical epithelium. Clusters were first observed in some animals as early as at 3 d.p.i. (diameter 150±28 µm, CD4 T cells per cluster 36±12, n=15). By 7 d.p.i., clusters were detected in all examined animals, and by 14 d.p.i. the sizes of clusters and densities of CD4 T cells in the clusters increased dramatically. C) Local expansion in vRNA+ cells predominantly maps to CD4 T cell foci. Based on the sizes of CD4 T cells clusters at 3 d.p.i. and their cell densities, we defined in this study the minimal cluster as at least 24 CD4 T cells (the minimal number of CD4 T cells in clusters at 3 d.p.i.) within a range of 178µm in diameter (the maximal diameter of clusters at 3 d.p.i.). With this definition, we quantified the percentage of vRNA+ cells that fell into a minimal CD4 T cell cluster.

FIGURE 3

The kinetics of accumulation of CD68⁺ (A) and CD163⁺ (B) macrophages and BDCA2⁺ (C) and CD123⁺ (D) pDCs in the cervical tissues over the course of vaginal infection. Macrophages and pDCs were temporally antecedent to CD4 T cell recruitment. Every dot represents an individual animal. At least 2–3 random cervical sections were stained and counted for every animal.

FIGURE 4

Co-localization in subjacent tissue sections of CD4⁺ T cells, macrophages, pDCs and vRNA+ cells (original magnification ×200) in the cervical transformation zone and adjoining endocervical tissues. Macrophages were defined as CD68+ and CD163+ cells; and pDCs as CD123+ and BDCA-2+ cells. (A–B) Macrophages and pDCs often co-cluster under the cervical epithelium, even prior to CD4 T cell recruitment. Representative images in each panel were obtained from 2 different animals (4–5 d.p.i.), in which CD4 recruitment was not yet detectable in the cervical tissues. (C–F) By 7 d.p.i., macrophages and pDCs were consistently found to co-localize in cervical CD4 T cell foci in all 8 examined animals. Representative images in every panel were obtained from 4 different animals. Adjacent sections were used in panels A–F. (G–H) vRNA⁺ cells (appear black in transmitted light) were primarily localized in regions with macrophages and pDCs. Representative images in each panel were obtained from 2 different animals (7 d.p.i.).

FIGURE 4

Co-localization in subjacent tissue sections of CD4⁺ T cells, macrophages, pDCs and vRNA+ cells (original magnification ×200) in the cervical transformation zone and adjoining endocervical tissues. Macrophages were defined as CD68+ and CD163+ cells; and pDCs as CD123+ and BDCA-2+ cells. (A–B) Macrophages and pDCs often co-cluster under the cervical epithelium, even prior to CD4 T cell recruitment. Representative images in each panel were obtained from 2 different animals (4–5 d.p.i.), in which CD4 recruitment was not yet detectable in the cervical tissues. (C–F) By 7 d.p.i., macrophages and pDCs were consistently found to co-localize in cervical CD4 T cell foci in all 8 examined animals. Representative images in every panel were obtained from 4 different animals. Adjacent sections were used in panels A–F. (G–H) vRNA⁺ cells (appear black in transmitted light) were primarily localized in regions with macrophages and pDCs. Representative images in each panel were obtained from 2 different animals (7 d.p.i.).

FIGURE 4

Co-localization in subjacent tissue sections of CD4⁺ T cells, macrophages, pDCs and vRNA+ cells (original magnification ×200) in the cervical transformation zone and adjoining endocervical tissues. Macrophages were defined as CD68+ and CD163+ cells; and pDCs as CD123+ and BDCA-2+ cells. (A–B) Macrophages and pDCs often co-cluster under the cervical epithelium, even prior to CD4 T cell recruitment. Representative images in each panel were obtained from 2 different animals (4–5 d.p.i.), in which CD4 recruitment was not yet detectable in the cervical tissues. (C–F) By 7 d.p.i., macrophages and pDCs were consistently found to co-localize in cervical CD4 T cell foci in all 8 examined animals. Representative images in every panel were obtained from 4 different animals. Adjacent sections were used in panels A–F. (G–H) vRNA⁺ cells (appear black in transmitted light) were primarily localized in regions with macrophages and pDCs. Representative images in each panel were obtained from 2 different animals (7 d.p.i.).

FIGURE 5

The kinetics of mast cell accumulation in the cervical tissues. There was a slight increase in mast cells in the cervical tissues at 7 d.p.i.

FIGURE 6

Early chemokine expression in cervical tissues after vaginal challenge. (A) CCL20, CXCL8, and CCL3 were induced in the endocervical epithelium within 24h after vaginal inoculation. Epithelial expression of these chemokines persisted through the course of infection examined in this study. By contrast, these chemokine ligands were not expressed in the cervical tissues of SIVmac239Δnef-vaccinated animals after SIV vaginal challenge. (Original magnification ×200) (B) Microarray analysis of cervical necropsy tissues showed that the transcriptional levels of CCL3, CCL20 and CXCL8 increased after vaginal infection in unvaccinated animals, but remained at basal levels in SIVmac239Δnef-vaccinated animals. (C) Quantitative image analysis (QIA): The expression of chemokines was quantified by measuring the intensities of pixel per µm² on IHC stained sections.

FIGURE 6

Early chemokine expression in cervical tissues after vaginal challenge. (A) CCL20, CXCL8, and CCL3 were induced in the endocervical epithelium within 24h after vaginal inoculation. Epithelial expression of these chemokines persisted through the course of infection examined in this study. By contrast, these chemokine ligands were not expressed in the cervical tissues of SIVmac239Δnef-vaccinated animals after SIV vaginal challenge. (Original magnification ×200) (B) Microarray analysis of cervical necropsy tissues showed that the transcriptional levels of CCL3, CCL20 and CXCL8 increased after vaginal infection in unvaccinated animals, but remained at basal levels in SIVmac239Δnef-vaccinated animals. (C) Quantitative image analysis (QIA): The expression of chemokines was quantified by measuring the intensities of pixel per µm² on IHC stained sections.

FIGURE 6

Early chemokine expression in cervical tissues after vaginal challenge. (A) CCL20, CXCL8, and CCL3 were induced in the endocervical epithelium within 24h after vaginal inoculation. Epithelial expression of these chemokines persisted through the course of infection examined in this study. By contrast, these chemokine ligands were not expressed in the cervical tissues of SIVmac239Δnef-vaccinated animals after SIV vaginal challenge. (Original magnification ×200) (B) Microarray analysis of cervical necropsy tissues showed that the transcriptional levels of CCL3, CCL20 and CXCL8 increased after vaginal infection in unvaccinated animals, but remained at basal levels in SIVmac239Δnef-vaccinated animals. (C) Quantitative image analysis (QIA): The expression of chemokines was quantified by measuring the intensities of pixel per µm² on IHC stained sections.

FIGURE 7

Profiles of chemokine ligands and receptors induced in cervical macrophages (A), pDCs (B), and CD4 T cells (C) relevant to the recruitment by epithelial expression of CCL20, CCL3, and CXCL8, the co-clustering, and the chemokine concentration gradient. (A) Cervical macrophages (CD68⁺) were CCR5⁺, CCR6⁺, CXCR1⁺, and CXCR2⁺ and hence could be recruited by their ligands expressed in epithelium. Cervical macrophages produced CCL3, CCL5, CXCL8 (IL-8), and CXCL10/IP-10, the latter consistent with recruitment of CXCR3⁺ pDCs and CD4 T cells (B) Cervical pDCs (BDCA2⁺ or CD123⁺) were CCR6⁺, CXCR1⁺, and CXCR3⁺; hence could be recruited by ligands expressed in epithelium and macrophages. The recruited pDCs produced CCL5 as well. (C) Cervical CD4 T cells were CCR6⁺, CXCR1⁺, CXCR2⁺ and CXC3R⁺; and expressed CCL4 and CCL5. The endocervical epithelium was also CXCL10 (A) and CCL4 positive (C) in some animals as shown in the figures, but we did not observe consistent expression of these chemokines in all examined animals. These montages were constructed with images with original magnification of ×200. Macrophages were profiled in cervical transformation zone and adjoining endocervical tissues from all animals at 3 d.p.i., pDCs at 3 d.p.i., and CD4 T cells at 7 d.p.i. Only the most representative images were shown here. Nuclei stained blue with TOTO-3. White dashed line marks the endocervical epithelium.

FIGURE 7

Profiles of chemokine ligands and receptors induced in cervical macrophages (A), pDCs (B), and CD4 T cells (C) relevant to the recruitment by epithelial expression of CCL20, CCL3, and CXCL8, the co-clustering, and the chemokine concentration gradient. (A) Cervical macrophages (CD68⁺) were CCR5⁺, CCR6⁺, CXCR1⁺, and CXCR2⁺ and hence could be recruited by their ligands expressed in epithelium. Cervical macrophages produced CCL3, CCL5, CXCL8 (IL-8), and CXCL10/IP-10, the latter consistent with recruitment of CXCR3⁺ pDCs and CD4 T cells (B) Cervical pDCs (BDCA2⁺ or CD123⁺) were CCR6⁺, CXCR1⁺, and CXCR3⁺; hence could be recruited by ligands expressed in epithelium and macrophages. The recruited pDCs produced CCL5 as well. (C) Cervical CD4 T cells were CCR6⁺, CXCR1⁺, CXCR2⁺ and CXC3R⁺; and expressed CCL4 and CCL5. The endocervical epithelium was also CXCL10 (A) and CCL4 positive (C) in some animals as shown in the figures, but we did not observe consistent expression of these chemokines in all examined animals. These montages were constructed with images with original magnification of ×200. Macrophages were profiled in cervical transformation zone and adjoining endocervical tissues from all animals at 3 d.p.i., pDCs at 3 d.p.i., and CD4 T cells at 7 d.p.i. Only the most representative images were shown here. Nuclei stained blue with TOTO-3. White dashed line marks the endocervical epithelium.

FIGURE 7

Profiles of chemokine ligands and receptors induced in cervical macrophages (A), pDCs (B), and CD4 T cells (C) relevant to the recruitment by epithelial expression of CCL20, CCL3, and CXCL8, the co-clustering, and the chemokine concentration gradient. (A) Cervical macrophages (CD68⁺) were CCR5⁺, CCR6⁺, CXCR1⁺, and CXCR2⁺ and hence could be recruited by their ligands expressed in epithelium. Cervical macrophages produced CCL3, CCL5, CXCL8 (IL-8), and CXCL10/IP-10, the latter consistent with recruitment of CXCR3⁺ pDCs and CD4 T cells (B) Cervical pDCs (BDCA2⁺ or CD123⁺) were CCR6⁺, CXCR1⁺, and CXCR3⁺; hence could be recruited by ligands expressed in epithelium and macrophages. The recruited pDCs produced CCL5 as well. (C) Cervical CD4 T cells were CCR6⁺, CXCR1⁺, CXCR2⁺ and CXC3R⁺; and expressed CCL4 and CCL5. The endocervical epithelium was also CXCL10 (A) and CCL4 positive (C) in some animals as shown in the figures, but we did not observe consistent expression of these chemokines in all examined animals. These montages were constructed with images with original magnification of ×200. Macrophages were profiled in cervical transformation zone and adjoining endocervical tissues from all animals at 3 d.p.i., pDCs at 3 d.p.i., and CD4 T cells at 7 d.p.i. Only the most representative images were shown here. Nuclei stained blue with TOTO-3. White dashed line marks the endocervical epithelium.

FIGURE 8

Clustering of the recruited cells in foci where chemokines CXCL8 and CCL3 are concentrated beneath the cervical epithelium close to the cervical transformation zone (left panels, encircled). Recruited CD4 T cells (red) predominantly co-localize to these foci of concentrated chemokines (right). The representative montages were obtained from cervical tissues from an animal at 7 d.p.i. These montages were constructed with images with original magnification of ×200. TZ: transformation zone; EctoCVX: ectocervix; EndoCVX: endocervix.

FIGURE 9

Model of the epithelium-innate immune cell axis and the sequential events in early mucosal responses that lead to CD4⁺ T cell recruitment. (1) Cervical epithelium responds to exposure to the SIV inoculum by producing CCL3, CCL20, and CXCL8; (2) the epithelial chemokines recruit macrophages and pDCs; (3) the macrophages and pDCs themselves produce CCL3, CCL5, CXCL8, and CXCL10 to create a focal concentrated source of chemokines beneath the epithelium; (4) this chemokine gradient recruits β-chemokine-producing CD4 T cells along with more macrophages and pDCs to generate a positive feed forward mechanism to sustain further increases in CD4 T cell targets.