Defining the Mechanisms of HIV Entry and Interactions with the Female Genital Tract

Transcription

1 Defining the Mechanisms of HIV Entry and Interactions with the Female Genital Tract Carias, A. M. HOPE LAB Northwestern University: Department of Cell and Molecular Biology

2 HIV Transmission HOW DOES HIV BREACH EPITHELIAL BARRIERS? Shattock & Moore, 2003

3 Female Genital Tract - Columnar Endocervix Single-cell barrier 10-30µm thickness Superficial target cells Mucus production Shattock & Moore, 2003

4 Female Genital Tract - Squamous Ectocervix Vagina Robust: From µm thick Superficial Langerhans cells Underlying target cells Multiple cell layers Shattock & Moore, 2003

5 The stratified squamous epithelium consists of 4 general layers Basal Layer Spinosa Layer Granulosa Layer Stratum Corneum

6 Visualizing HIV in Tissue HIV in explants 430nm

7 Explant cultures surgery Explant: Ectocervix and Endocervix for single donor (premenopausal hysterectomy). Dissect into individual sections 1cm 3 thick. Freeze samples down in OCT. Section. Image and measure distance to nearest epithelial surface. Amount: ng/ml p24 Time: Incubate virus 4 hours with explant.

8 HIV can breach columnar barriers in the endocervix Lumen Lamina Propria 15 Nuclei Nuclei Pre photoactivation Cytokeratin 7 Post photoactivation

9 Virus Penetration Depths of Individual Samples (Endocervix) Ba L Yu Bru3 BruD Cervix Donors

10 HIV can breach the squamous epithelium Most virions appears to move interstitially (~90% of time) DAPI Pre Photoactivation WGA Post Photoactivation 15

11 Virus Penetration Depths of Individual Samples (Ectocervix) Ba L Yu Bru3 BruD Cervix Donors

12 Ectocervix vs. Endocervix ECTO (22)ENDO (16) Analysis of tissue from ~30 donors

13 Rhesus Macaques 11/2/2009

14 Virus can enter the stratified squamous epithelium of the macaque Ectocervix and vagina contain many virions Virions appears to move interstitially (~50% of time) Virion penetration mimics human explant system 15

15 Human cervical explants vs. in vivo rhesus macaques HUMAN CERVIX (5 donors) RHESUS MACAQUE (5 animals)

16 EXPLANT EXPLANT MACAQUE MACAQUES Rhesus Macaque Explant data correlates with Human Cervical Explant data Results are similar to those illustrated in human cervical explants More virions associated with endocervix than seen in in vivo macaque studies EXPLANT HUMANS

17 Nuclei E cadherin Background Analysis of proteins in cellular junctions 30 Modified from R. Shattock

18 Breakdown of intercellular contacts Loss of tight junctions and adherens near lumen Roughly corresponds to viral penetration Nuclei Claudin 4 background 30

19 Observed correlation of Fluid phase marker with cell junctions Bovine serum albumin Marker of where water can enter the tissue Fluid phase marker penetrates only where cell junctions are compromised DAPI E cadherin BSA 30

20 Penetration of BSA in Macaque Vagina 40X Nuclei WGA BSA Illustrates heterogeneity in tissue structure 4 hour in vivo incubation Macaque vagina 40

21 Does virus entry correlate with fluid phase marker? 60X Nuclei WGA BSA 30

22 60X Nuclei WGA BSA 30 11/2/2009

23 100X DAPI Pre Activation Post Activation

24 10x more virus penetrates In regions where BSA enters greater than 5 µm <5µM >5µM Z SCANS VIRIONS PENETRATING VIRIONS VIRUS/Z SCAN PENETRATING VIRUS/Z SCAN AVG PENETRATING VIRUS DEPTH MAX DEPTH BSA < 5um um 29.33um BSA > 5um um 34.07um

25 Role of HIV envelope in penetration into cervical explants Medium penetration the same for R5, X4, or no envelope into ectocervix consistent with diffusion

26 Target cells and HIV penetration DAPI CD1a E caderin 40

27 Conclusions HIV can penetrate the intact female genital epithelia of humans and Macaques. HIV appears to move interstitially Virus penetration correlates with BSA depths HIV can enter tissue to depths where target cells (LC dendrites) exists.

28 Acknowledgements Thomas J. Hope HOPE LAB Scott McCoombe Mike McRaven Shetha Shukair Minh Dinh Nicole Byers Meegan Anderson Jill Kowalski Z Kelley Kelly Fahrbach Anna Figueiredo Sheila Barry Dan Gallo Cindy Danielson Amy Hulme Ann Koons Shannon Allen Ayanna Flegler Zia Okocha Doug Dylla Katharina Rothwangl Gianguido Cianci Chris Rold Collaborators Ron Veazey Robin Shattock

29 Columnar vs. Squamous: surface area and access 11/2/2009

30 Columnar vs. Squamous: surface area and access 11/2/2009

31 Columnar vs. Squamous: surface area and access 11/2/2009