Upon viral infection, assessing the host nasal epigenome

In this case, they used not pooled samples but individual samples to ensure they did not have a driver individual in their pools who might have been driving the influenza B signature. They used the full populations of hundreds of samples from the infants, and they “noticed a top and significant association where influenza B samples continued to be the strongest associations shown by cytokines and chemokines that are known to be the first responders in viral infections and attract the immune cells.”

When they went back to their DNA methylation data and looked at the magnitude, creating differentially methylated regions (DMR) and contrasting the negative controls, “the influenza B continued to be the largest epigenome landscape change,” at more than 14,000 DMRs, she said. Influenza A and parainfluenza were much lower in frequency.

They also assessed whether this change-up in the viral infection was an activation or a repression and whether it became hypo- or hypermethylated. They found a bimodal pattern. “We assumed that the majority will be hypomethylated, something that becomes activated,” Dr. Grundberg said, because if there is viral infection, “we need an activation of gene machinery.” While activation was a large component, “an interesting component was hypermethylation.”

In examining the hypermethylated regions, “we were intrigued by seeing how significantly enriched they were among immune regulatory elements,” Dr. Grundberg said. “So basically, something becomes suppressed or deactivated in the immune cells, which continues to be intriguing.”

Many different immune cells are present in the nasal mucosa. When they zoomed in and mapped it further, they were able to identify those regulatory elements that are specific to the myeloid lineage and specifically to the neutrophils and monocytes that become deactivated, she said.

To assess the robustness of the influenza B infection epigenome perturbation, Dr. Grundberg’s group returned to the same 2018–2019 viral season and redid the pooling strategy and age-matched samples, repeating it for viruses that showed the most striking effect. They validated the significant regions seen in their discovery, “and again influenza B was shown to be extremely robust,” she said. “We could validate 50 percent of our differentially methylated regions in an independent population.” The replication rate was consistent for activated or deactivated regions. Other viral infections showed poor replication rates (influenza A, 12 percent; rhinovirus, three percent; human metapneumovirus, two percent), “potentially indicating we don’t have a robust impact on the host epigenome by these viruses potentially linked to milder respiratory symptoms.”

Further analysis of the influenza B hypomethylated signatures revealed links to epithelial-specific genes, Dr. Grundberg said. “We performed a gene ontology analysis and could identify expected genes that got activated,” those in interferon (e.g. CX3CL1, CX3CR1), including the type three interferon-related genes (IL 10RB), as well as key antiviral-related genes (e.g. ISG15, ISG20). The most relevant and “extremely striking” link, she said, was to bronchial epithelial cells.

The group tried to assess other ways of replicating its findings that influenza B hypomethylated signatures are linked to upregulated genes in epithelial cells. “We’ve been doing protein analysis, methylation analysis, but we’ve also shown that we can use a similar type of sample for single-cell analysis. Here we need to be a little quicker when we do the analysis, work more closely with the clinical labs,” she said.

They obtained influenza B-positive samples and controls from the same season, performed single-cell analysis on them, and “were excited to see that we can map a lot of these genes that have been shown to be specific to the nasal mucosa,” Dr. Grundberg said. When they overlapped the patterns with their activated genes or regulatory elements for epigenome analysis, “they validated quite well.”

The influenza B hypermethylation signatures were “not trivial,” she said. The authors of a 2021 article used a comparable approach but focused on vaccinations and single-cell and epigenome assessments using blood (Wimmers F, et al. Cell. 2021;​184[15]:3915–3935.e21). Their findings, she said, were similar to those of her group: a reduction in promising accessibility and activation but also a fair amount of deactivation. “They speculate that that could potentially be to avoid excess inflammatory host damage during late stages of infection. That’s the leading hypothesis we see on the epigenome analysis as well.”

But Dr. Grundberg and her colleagues were interested also in the age-dependent effect. They were working with infants so they repeated their analysis but expanded their age group up to three years. “We then started to see a completely different pattern—this clear methylation was only seen in the first few months of life,” she said. Hypermethylation response to influenza B infection peaked between ages two and four months, declining until eight months, then leveling out likely due to vaccinations having been introduced.

Age association of viral infection then became “a side story” for the project, Dr. Grundberg said, so her group set up a supporting study to see how the nasal mucosa cell proportions change across ages. “We created a cell atlas across the lifespan where we focused on the healthy mucosa or being negative in the pathogen testing.” Children’s Mercy Kansas City conducts respiratory testing for employees, so they were able to expand the nasal swab sampling up to age 60. Their single-cell analysis on the extended age range of nasal samples confirmed the presence of “a rich epithelial and immune landscape,” she said.

The immune cell occupancy in normal nasal mucosa declines with age when comparing children with adults. If looking only at children, it was higher in older children—peaking at age two—compared with infants. The results were in line with influenza being dangerous for the very young and very old.

While finalizing their analysis in spring 2020, Dr. Grundberg and colleagues incorporated COVID-19 samples. They repeated the epigenome analysis using nasal samples collected from children with relatively mild SARS-CoV-2 infection. “If we thought that influenza B had been an outlier, COVID-19 was in a different level,” she said—reaching nearly 80,000 DMRs, a striking epi­genome perturbation.

“We incorporated age in a similar way we did with influenza B associations, but as this was before any sort of COVID-19 vaccination was introduced, we could see a very different pattern,” Dr. Grundberg said. All those assessed from age four weeks to 19 years had a similar effect in the epi­genome, apart from one case that dropped and was clustered close to the negatives, “potentially an asymptomatic case,” she said, noting the high volume of preoperative testing performed at the time. “Unfortunately, we don’t have that clinical information.”

Her group has set up new programs to study individual samples and perform targeted approaches, “given the significant cost of whole genome bisulfite sequencing,” she said. In one study of 60 adults in spring 2020 using salvage nasal samples, “we had relatively rich clinical information so we could distinguish those who are hospitalized versus those who are mild and do similar epigenetic assessments.” They found that FUT4 hypermethylation—the CD15 marker of neutrophils—is linked to COVID-19 severity.

“We are excited that salvage nasal swab sampling has been successful” for high-resolution epigenome, proteome, and single-cell expression analysis, she concluded. In using the salvage samples, they can identify epigenetic signatures that seem to distinguish viral infections and potentially infection severity.

If these types of epigenome analyses are done across individuals, as shown in SARS-CoV-2 infection, she said, “we may be able to start to inform about markers for disease severity.”

Amy Carpenter Aquino is CAP TODAY senior editor.