key: cord-0735802-kdegnr6i
authors: Trevelin, Silvia C.; Pickering, Suzanne; Todd, Katrina; Bishop, Cynthia; Pitcher, Michael; Mesa, Jose Garrido; Montorsi, Lucia; Spada, Filomena; Petrov, Nedyalko; Green, Anna; Shankar-Hari, Manu; Neil, Stuart J.D.; Spencer, Jo
title: Disrupted Peyer’s patch microanatomy in COVID-19 including germinal centre atrophy independent of local virus
date: 2021-12-20
journal: bioRxiv
DOI: 10.1101/2021.12.17.473179
sha: d445e8d9f4aef4c4fb826f7e02f4df03983b300b
doc_id: 735802
cord_uid: kdegnr6i

Confirmed SARS-coronavirus-2 infection with gastrointestinal symptoms and changes in microbiota associated with coronavirus disease 2019 (COVID-19) severity have been previously reported, but the disease impact on the architecture and cellularity of ileal Peyer’s patches (PP) remains unknown. Here we analysed post-mortem tissues from throughout the gastrointestinal (GI) tract of patients who died with COVID-19. When virus was detected by PCR in the GI tract, immunohistochemistry identified virus in epithelium and lamina propria macrophages, but not in lymphoid tissues. Immunohistochemistry and imaging mass cytometry (IMC) analysis of ileal PP revealed depletion of germinal centres (GC), disruption of B cell/T cell zonation and decreased potential B and T cell interaction and lower nuclear density in COVID-19 patients. This occurred independent of the local viral levels. The changes in PP demonstrate that the ability to mount an intestinal immune response is compromised in severe COVID-19, which could contribute to observed dysbiosis.

Dysregulated immune response to infection with SARS-coronavirus-2 (SARS-CoV-2) is the 44 main driver of mortality in coronavirus disease 2019 (COVID-19) (1, 2). Whilst respiratory 45 dysfunction is common, symptoms involving the gastrointestinal (GI) tract has been identified, 46 including vomiting and diarrhoea in 12% of the patients (3). Moreover, viral RNA has been 47 found in stool samples (4) and viral particles identified in ileal epithelium (5). The receptor 48

for SARS-CoV-2 angiotensin converting enzyme 2 (ACE2) is expressed on the luminal surface 49 of epithelial cells throughout the GI tract. It has been proposed that reservoirs of virus in the 50 GI tract could support longer lived antibody responses that are fundamental for controlling 51 virus replication or could be associated with persistent disease if ineffective (5, 6). However, 52 the consequences of SARS-CoV-2 infection on the GI immune system and the local ability to 53 respond to viral infection in severe disease is currently unknown. 54

The intestinal immune system is highly compartmentalised (7). Immune responses can 55 be initiated in gut-associated lymphoid tissue (GALT) (8). Activated B and T cells generated 56 in GALT acquire specific receptors, such as a4b7, CCR9 and CCR10 that allow them to home 57 to lamina propria following circulation via lymphatics and the blood (9-11). 58

Peyer's patches (PP) are clusters of GALT concentrated in the terminal ileum. A 59 common feature of PP from early life in humans is the presence of germinal centre (GC) that 60 are acquired in response to particulate antigens sampled from the gut lumen. The ensuing GC 61 response generates lamina propria plasma cells secreting IgA that is transported into the gut 62 lumen and that subsequently regulates the microbiota and maintains homeostasis (7, 12). 63 GC responses are regulated in part by transcription factor BCL6 (B-cell lymphoma 6) 64 that is considered a marker for GC cells. It is known that GCs can be lost in lymph nodes and 65 spleen in acute COVID-19, and this has been linked to diminishing of BCL6 + B and T cells in 66 these tissues and blood (6). Whether GALT is similarly impacted is not known. 67

Here, the virus was quantified and localised in samples of gastrointestinal tract from 68 patients who died with COVID-19 using reverse transcription quantitative PCR (RT-qPCR) 69 and immunohistochemistry. By immunohistochemistry and imaging mass cytometry (IMC), 70 the architecture and cellularity of PPs in the same samples were then explored in detail. 71 72

Identification of SARS-CoV-2 in tissue samples along the GI tract 74

We first quantified and localised SARS-CoV-2 in formalin-fixed paraffin embedded (FFPE) 75 samples of oesophagus, stomach, duodenum, ileum, colon, lungs and spleen from 7 males and 76 2 females who died after being diagnosed with COVID-19 (Supplementary Table 1 ). RT-77 qPCR analysis of N1 of SARS-CoV-2 nucleocapsid standardised to RNAse P revealed traces 78 of the virus in most tissues from COVID-19 patients but not controls ( Figure 1A In order to investigate better the architecture of PPs, ileal samples were initially double stained 88 with anti-CD45RB that is expressed by T and the B cells on the periphery of lymphoid tissues, 89 but not GC B cells (13) and anti-CD10 that stains the GC (14). The CD10:CD45RB ratio was 90 significantly reduced in COVID-19 patients compared to controls, irrespective of the local 91 levels of viral RNA measured by RT-qPCR (Figure 2A Figure 3E ), although the percentages of CD14 + CD16or CD14 + CD16 + or CD14 lo CD16 + 118 macrophages were similar between groups ( Figure 3F ). 119

The area of the ablated regions occupied by the lymphoid tissue was comparable 120 between COVID-19 samples and controls (0.08±0.028 vs 0.09±0.021 mm 2 ). However, the 121 cellular density was significantly reduced in lymphoid tissue in COVID-19 ( Figure 4A ). The decreased cell density of the PP and depletion of the GC in ileal follicles of patients 178 with COVID-19 is consistent with impaired T and B cell interaction, which could contribute 179 to failure to generate a long-term response to local antigens and contribute to dysbiosis (19, 180 30). 181

In conclusion, patients with severe COVID-19 show significant impaired architecture 182 and cellularity of PP. The resulting poor local immunity could contribute to dysbiosis. Our 183 findings also suggest that oral vaccination to prevent COVID-19 disease could not be effective 184 if patients were already ill, since the gut immune system is compromised with features 185 indicating that they would lack the ability to mount an efficient immune response. 

Imaging Mass Cytometric 287 Analysis of Postmortem Tissues Reveals Dysregulated Immune Cell and Cytokine 288 Responses in Multiple Organs of COVID-19 Patients

Prevalence of Gastrointestinal Symptoms and Fecal Viral Shedding in Patients With 295 Coronavirus Disease 2019: A Systematic Review and Meta-analysis

Virological assessment of hospitalized patients with COVID-2019

Evolution 301 of antibody immunity to SARS-CoV-2

Loss 303 of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19

Human 306 gut-associated lymphoid tissues (GALT); diversity, structure, and function

Intestinal mucosal barrier function in health and disease

Alpha 4 311 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin 312 MAdCAM-1

A Subset of CCL25-314 Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer

Critical roles of chemokine receptor CCR10 317 in regulating memory IgA responses in intestines

Form follows function: lymphoid tissue 320 microarchitecture in antimicrobial immune defence

Pivotal advance: 323 CD45RB glycosylation is specifically regulated during human peripheral B cell 324 differentiation

326 Comparison of germinal center markers CD10, BCL6 and human germinal center-327 associated lymphoma (HGAL) in follicular lymphomas

329 histoCAT: analysis of cell phenotypes and interactions in multiplex image cytometry 330 data

ilastik: 332 interactive machine learning for (bio)image analysis

CellProfiler: image analysis software for identifying and quantifying cell phenotypes

Data is shown as mean± SEM. (n=4 controls and 5 458 patients). Kruskal-Wallis followed Dun's post

Enhanced relative numbers of macrophages in ileal follicles in Peyer's patches 461 (PP) of COVID-19 patients

CD4 + FoxP3 + T cells in ileal Peyer's Patch (PP) from COVID-19 -and COVID-19 + patients

Representative images from histoCAT showing CD4 (green), CD8a (blue)

CD14 (green) and CD16 (magenta) signals in PPs on the left, and mean CD68 signals 466 on the right. (F) Percentages of follicular CD14 + , CD16 + and CD14 + CD16 + cells from CD68 + 467 cell population. Data is shown as mean± SEM

Dun's post-test in D and two tailed Mann-Whitney t test in E. *P<0

Schematic depicting the microanatomical features identified in ileal post mortem 495 samples from patients who died with COVID-19: depletion of the germinal centre (GC

Peyer's patches, enhanced numbers of follicular macrophages

fewer CD27 + memory B cells and lower expression CD74 on B cells