The spatial biology of HIV infection

Abstract

HIV infection implicates a spectrum of tissues in the human body starting with viral transmission in the anogenital tract and subsequently persisting in lymphoid tissues and brain. Though studies using isolated cells have contributed significantly towards our understanding of HIV infection, the tissue microenvironment is characterised by a complex interplay of a range of factors, all of which can influence the course of infection but are otherwise missed in ex vivo studies. To address this knowledge gap, it is necessary to investigate the dynamics of infection and the host immune response in situ using imaging-based approaches. Over the last decade, emerging imaging techniques have continually redefined the limits of detection, both in terms of the scope and the scale of the targets. In doing so, this has opened up new questions that can be answered by in situ studies. This review discusses the high-dimensional imaging modalities that are now available and their application towards understanding the spatial biology of HIV infection.

Fig 1. Schematic illustrating mucosal anogenital tissue compartments and the APCs they contain in both steady state and inflammation.

The Type II epithelial layer of the vagina, ectocervix, inner foreskin, fossa naviculars and anal canal consists of a multilayered stratified squamous epithelium (SSE) of keratinocytes whereas the Type I mucosa of the colorectum consists of thin and fragile monolayer of columnar epithelial cells. Underlying both epithelial surfaces is a layer of connective tissue referred to as lamina propria. The colorectum contains thin muscle layer called the muscularis mucosa which separates the lamina propria from the deeper submucosa. This tissue also contains lymphoid follicles which can span the lamina propria and submucosa. A) Contained within the SEE of the Type II mucosa are both LCs and epi-DCs, but in contrast to the SSE of skin, DCs predominate. Contained within the lamina propria of both Type I and II mucosal tissues are DC1, DC2, MDDC and macrophages. Although the submucosa of the colorectum contains some DCs, macrophages are the predominant myeloid subset. B) Inflamed anogenital tissues are associated with increased HIV transmission for which there are three potential reasons: 1) these tissues contain additional inflammatory APCs including pDC and new discovered DC3 and ASDC which express higher levels of the HIV entry receptor CCR5; 2) inflamed tissues result in a more fragile epithelium which is more susceptible to mucosal abrasions allowing the virus to cross the epithelial barrier; 3) anogenital inflammation is often associated with an abnormal microbiome which secrete metabolites which break down PrEP drugs.

Fig 2. RNAScope detection of HIV_Bal (red) interacting with CD11c⁺ dendritic cells (green) and CD4⁺ T cells (blue).

Images sourced from original publication [24].

Fig 3. Representative fluorescent image of rectum mucosa stained with DAPI (blue) and pan cytokeratin (green).

Illustrations of three spatial transcriptomic groups: A) Region of interest (ROI) spatial RNA sequencing (GeoMx), B) Spatial RNA sequencing (Visium, VisiumHD, Slide-SeqV2, StereoSeq), and C) imaging in situ hybridisation (CosMx, Xenium, Merscope). Created with Biorender.com.

Fig 4. Image processing steps and types of spatial analyses associated with high dimensional imaging datasets.

*Registration of images is a processing step that is specific to iterative fluorescence-based modalities such as CyCIF, CODEX and SABRE due to possible slide misalignments during image acquisition with every cycle. Created with Biorender.com.

Fig 5. Imaging mass cytometry of intraepidermal nerve fibres.

A) Representative image of cutaneous nerve fibres visualised using imaging mass cytometry (IMC). An indirect immunolabelling technique for PGP9.5 (green) and Class III β-Tubulin (red) combined with heavy metal-isotype conjugated tertiary antibodies was used to detect double-positive (PGP9.5⁺ β-Tubulin⁺, yellow) nerve fibres and to distinguish neuronal tissue from melanocytes, which also express Class III β-Tubulin, in the basal epidermis. An intraepidermal nerve fibre (white arrow, right) exhibiting a typical punctate morphology is seen extending from the stratum basale to the stratum granulosum. A thicker dermal nerve (white arrow, left) located within a dermal papilla is also visible. A, Inset) Representative image of intraepidermal nerve fibres (white arrowheads) detected by chromogenic immunohistochemistry for PGP9.5 (brown). B, C) Representative images of cutaneous nerve fibres and Langerhans cells visualised using IMC. Single-labelled (β-Tubulin⁺) intraepidermal nerve fibres (white arrowheads) are present in the stratum spinosum and stratum granulosum. Profiles of nerve fibres in the papillary dermis are also seen (not indicated). The Langerhans cells (white arrows) present in the epidermis are in close proximity to intraepidermal nerve fibres. For all IMC experiments, Zamboni’s-fixed, paraffin-embedded 3 mm skin punch biopsies were sectioned at 7μm and immunolabelled with heavy metal isotope-conjugated antibodies targeting proteins of interest (or tertiary antibodies in the case of indirect staining). Cell nuclei were labelled with Cell-ID Intercalator-Ir (Standard BioTools) and are indicated in blue in all three images. Acquisition was performed on a Hyperion+ Imaging System (Standard BioTools) and data was visualised in MCD Viewer (Standard BioTools). Abbreviations: IMC, Imaging Mass Cytometry; PGP9.5, Protein Gene Product 9.5; β-Tubulin, Class III β-Tubulin.