Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques

Abstract

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

SciScore for 10.1101/2021.09.08.459430: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: Ethics and biosafety statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Rocky Mountain Laboratories, NIH and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility, according to the institution’s guidelines for animal use, following the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Sex as a biological variable	not detected.
Randomization	Sample processing for organic and lipid mediator extraction: Sample order was randomized throughout each extraction.
Blinding	not detected.
Power Analysis	After de-waxing, antigen retrieval was performed using AR6 buffer (Akoya Biosciences) in a standard microwave with 45 seconds at 100% power and 15 minutes at 10% power.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
A mixture of anti-Mouse + anti-Rabbit HRP-conjugated secondary antibodies (Akoya Biosciences) was added for 10 minutes, then washed in TBST.	anti-Mouse + anti-Rabbit HRP-conjugated secondary antibodies suggested: None anti-Rabbit suggested: None
T-cell phenotyping of PBMC and lung cell samples: The T-cell surface staining cocktail contained the following antibodies: CD3 BV650, CD4 BUV395, CD8 PerCP-Cy5.5, CD28 BUV661, CD38 FITC, CD69 AF700, CD25 BUV496, CD154 BV605, HLA-DR APC-Cy7, CD45RA BUV563, CCR7 BV510, CD95 BUV737, CD49d PE-Cy7, CTLA4 BV785	CD4 suggested: (RayBiotech Cat# CS-11-0132, RRID:AB_1228050) CD28 suggested: (BD Biosciences Cat# 749971, RRID:AB_2874198) CD69 suggested: (BD Biosciences Cat# 558063, RRID:AB_2275535) CD25 suggested: (BD Biosciences Cat# 741144, RRID:AB_2870722) CD154 suggested: (BD Biosciences Cat# 748984, RRID:AB_2873384) CD45RA suggested: (BioLegend Cat# 304139, RRID:AB_2561369) CD95 suggested: (BioLegend Cat# 305646, RRID:AB_2629742) CTLA4 suggested: (BioLegend Cat# 369624, RRID:AB_2810582)
B-cell phenotyping of PBMC samples: B-cell surface staining was carried out using the following antibodies: CD3 BV650, CD27 BUV805, CD38 FITC, CXCR3 BV421, CD19 PE, CD20 BV510, CD21 BV786	CD27 suggested: (BD Biosciences Cat# 748704, RRID:AB_2873108) CXCR3 suggested: (BD Biosciences Cat# 741005, RRID:AB_2740628) CD20 suggested: (BD Biosciences Cat# 743611, RRID:AB_2741622) CD21 suggested: (BD Biosciences Cat# 742764, RRID:AB_2741029)
The above cocktails also contained CD28 BUV661 and CD49d antibodies (each at 1 μg/mL), as well as 0.67 μg/mL of GolgiStop and 1 μg/mL of GolgiPlug (BD).	CD49d suggested: None
Surface markers were stained for with a cocktail of the following antibodies: CD3 BV650, CD4 BUV395, CD8 PerCP-Cy5.5, CD95 BUV 737, CD45RA BUV563, CCR7 BV510, CD14 PE-Dazzle594, and CD16 PE-CF594.	CD3 suggested: (RayBiotech Cat# CS-11-0110, RRID:AB_1228004) CCR7 suggested: (BD Biosciences Cat# 563449, RRID:AB_2738212) CD14 suggested: (BioLegend Cat# 348805, RRID:AB_2889063) PE-Dazzle594 suggested: None CD16 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
VeroE6 cells were maintained in DMEM supplemented with 10% fetal calf serum, 1 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
Software and Algorithms
Sentences	Resources
Quantification of images such as selection of Ki67+ nuclei, segmentation of DAPI signal, identification of cell types, and quantification of BALT structures was performed in Imaris V9.5.0.	Imaris suggested: (Imaris, RRID:SCR_007370)
The 10X Genomics version 3.1 chemistry was used to generate barcoded cDNA and to generate final libraries according to the manufacturer’s protocol.	Genomics suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)
FACSDivaTM version 8.0.1 software.	FACSDivaTM suggested: None
Subsequently, t-SNE analysis60 was performed in the FlowJo (Treestar, version 10.7) environment on compensated parameters (nearest neighbor = 15, minimum distance = 0.5, iterations = 1000), FlowSOM (version 2.9)24 analysis was conducted using compensated parameters and fixed generation of 15 meta clusters (set speed = 3)60.	FlowJo suggested: (FlowJo, RRID:SCR_008520) FlowSOM suggested: (FlowSOM, RRID:SCR_016899)
Statistics and data analysis: Most statistical analyses were performed in GraphPad V9.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
RNA sequencing data have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE183579.	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Despite these limitations, the utilization of a large set of high-parameter techniques in one study to generate a time-resolved, multi-site profile of virus-induced innate and adaptive immune responses, has resulted in a comprehensive characterization of SARS-CoV-2 infection in the rhesus macaque model. Importantly, our findings highlight essential differences and similarities in the immune response to SARS-CoV-2 infection between rhesus macaques and humans that may be affected by age. As such, our study provides new insights into the age-related immune dynamics of SARS-CoV-2 infection and represents a substantial advance in available models of age-associated changes in immunity.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques

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