RNA Sequencing Analysis of Peripheral Blood Mononuclear Cells Reveals a Novel Pathway Associated with COVID-19 Deterioration

The severity of COVID-19 depends on the host immune cell response. Most patients with COVID-19 had mild disease, but up to 30% of patients developed a severe disease. Risk factors associated with severe diseases such as age, smoking, and comorbidities are known, while molecular signatures that predict the disease progression are still emerging. Here, we investigated gene expression pro�les in peripheral blood mononuclear cells (PBMCs) from patients with COVID-19 using RNA-sequencing analysis (RNA-seq) based on the hypothesis that deteriorating patients have a unique molecular signature in PBMCs at an early stage of the disease. Twelve patients were included in RNA-seq (deteriorated vs. nondeteriorated, each n = 6). RNA-seq demonstrated 156 differentially expressed genes, and pathway analysis showed that “Nucleosome assembly” was the most important pathway, with 24 HIST1 cluster genes upregulated in the deteriorated group. In silico prediction of upstream transcription factors exhibited protein inhibitors of activated STAT 4 (PIAS4) as the most signi�cant. TP53, STAT1, and IKBKG associated with COVID-19 pathophysiology are predicted to interact with PIAS4. Our results show that elevated HIST1 cluster gene expression predicts COVID-19 deterioration, which may induce histone-mediated lung injury and coagulopathy. The molecular mechanism could involve PIAS4-mediated transcriptional regulation and in�ammatory signal transduction.

The severity of COVID-19 depends on the host immune cell response and dysregulation 5 .Risk factors associated with severe diseases such as age, cancer, chronic obstructive pulmonary disease, chronic kidney disease, diabetes mellitus, hypertension, hyperlipemia, obesity, and smoking are well known 6,7 .Also, laboratory test ndings such as lymphopenia and thrombocytopenia and elevated D-dimer, Creactive protein (CRP), and lactate dehydrogenase (LDH) may be associated with severe respiratory failure and death 6 .Nevertheless, molecular signatures related to the dysregulation of the immune response and the disease deterioration are not fully uncovered.
The rst line of host defense against viral infection relies on the innate immune system 5,8 in which cells such as monocytes, dendric cells, natural killer cells, and neutrophils are involved.In response to pathogen-associated molecular patterns derived from viruses, innate immune cells activate in ammatory signaling and cytokine production via pattern recognition receptors (PRRs) on the cell surface 8 .PRRs typi ed by Toll-like receptors (TLRs) also recognize damage-associated molecular patterns (DAMPs), endogenous molecules, including nucleic acids, HMGB1, heat shock proteins, S100 proteins, and histones, and amplify in ammatory signaling 9 .These events in immune cells are coordinated to promote in uenza-induced lung injury 10 and are suggested to play a critical role in COVID-19 11 ; however, little is known about speci c molecules and mechanisms related to disease progression and deterioration.
Based on this background, we reasoned that a comprehensive search for genes dysregulated in peripheral blood mononuclear cells (PBMCs) could improve our understanding of the mechanisms underlying immune response dysfunction.Since PBMCs include the gatekeeper immune cells of viruses spread during systemic viral infections and several subpopulations that may cooperate in the immune response 12 , this might facilitate the development of biomarkers and targeted therapies in COVID-19.We, therefore, applied RNA-sequencing analysis (RNA-seq) to investigate gene expression pro les of PBMCs derived from patients with COVID-19.PBMCs were collected from early-stage nonsevere patients with COVID-19 and analyzed by grouping patients into two groups: deteriorated and nondeteriorated, based on the subsequent clinical courses, to explore the pro le predictive of COVID-19 deterioration.RNA-seq revealed 156 differentially expressed genes (DEGs) between groups.Pathway analysis of the DEGs exhibits "Nucleosome assembly" as the top pathway, including HIST1 cluster genes that are mostly upregulated in the deteriorated group.In silico analysis demonstrated that protein inhibitors of activated STAT 4 (PIAS4) could be a possible upstream regulator in gene expression and in ammatory response by interacting with TP53, STAT1, and IKBKG.

Results
Baseline characteristics of patients with COVID-19 at PBMCs collection PBMCs were collected from early-stage nonsevere patients with COVID-19 (Median 4 days, range 3-6 days after symptom onset).Based on the subsequent clinical courses, patients were grouped into two groups, deteriorated and nondeteriorated, and PBMCs from deteriorated (n = 6) and nondeteriorated (n = 6) patients were analyzed by RNA-seq (Figure 1a).There were no signi cant differences in clinical characteristics such as body temperature, pulse rate, and oxygen saturation between the groups at sample collection.Still, laboratory testing showed higher CRP and LDH in the deteriorated group compared with the nondeteriorated group (Table 1).

RNA-seq of PBMCs and validation with reverse-transcribed quantitative polymerase chain reaction (RT-qPCR)
RNA-seq was performed on RNA isolated from PBMCs under deteriorated versus nondeteriorated groups (Fig. 1a) and revealed 156 DEGs between groups analyzed by DESeq2.Among 156 DEGs, 102 were upregulated, and 54 were downregulated in the deteriorated group compared with the nondeteriorated group (Fig. 1b, Fig. S1, Table S1).We also validated gene expression of HIST1H2BO and HIST1H2AE, the top two statistically signi cant genes, by RT-qPCR analysis and con rmed that those genes were differentially expressed with statistical signi cance (Fig. 1c).
Functional Enrichment analysis based on the DEGs Functional enrichment analysis based on the 156 DEGs were performed to explore pathways related to COVID-19 deterioration.Signi cant enrichment was observed in pathways related to nucleosome or chromatin assembly, including terms such as "Nucleosome assembly," "Nucleosome organization," and "DNA packaging" (Fig. 2a).Pathways related to immune response such as "Innate immune response in mucosa" and "Organ or tissue speci c immune response" were also shown but with less signi cance.Gene set enrichment analysis (GSEA) also demonstrated "Nucleosome assembly" as the top pathway, and the pathway included 24 HIST1 cluster genes upregulated in the deteriorated group (Fig. 2b).No term regarding cellular proliferation or apoptosis is shown.Thus, the results indicate that PBMCs from deteriorating COVID-19 heightened HIST1 cluster gene transcription more than genes related to proin ammatory response at the early stage of disease, which may result in histone-induced lung injury.

In silico predictions of upstream transcription factors associated with COVID-19 deterioration
We next explored upstream transcription factors that induced the differential gene expression in PBMCs associated with COVID-19 deterioration using GSEA.We found four transcription factors of signi cance, false discovery rate below 0.05 (Fig. 3a).Among those factors, PIAS4 exhibited the highest enrichment score, including multiple HIST1 cluster genes as core enrichment genes (Fig. 3b).PIAS4 is an E3-type small ubiquitin-related modi er (SUMO) ligase and can modify the function of signal transducers or transcription factors essential for the immune response to viral infection 13,14 .In line with this, we focused on PIAS4, explored its interaction partners using the STRING database, and found that PIAS4 interacts strongly and physically with signal transducers and transcription factors associated with COVID-19 pathogenesis, including TP53, STAT1, and IKBKG 15,16 (Fig. 4).Thus, our in silico analysis indicates that PIAS4 is an essential factor in the pathogenesis of COVID-19 and disease progression, possibly through modifying the in ammatory response and HIST1 cluster gene expression.

Discussion
In this study, we identi ed a PBMCs gene set differentially expressed between deteriorated and nondeteriorated COVID-19 at the early stage of the disease.The gene set is highly enriched in HIST1 cluster genes, mostly upregulated in severe diseases before disease deterioration.Based on the gene set, we demonstrate that PIAS4 could be an upstream regulator of HIST1 cluster gene transcription and a modulator of an in ammatory response by interacting with in ammatory signaling molecules.Our results show that dysregulated PIAS4 function and HIST1 cluster gene expression in PBMCs could contribute to COVID-19 exacerbation.
Histone proteins are intranuclear proteins that form nucleosomes.They play key roles in DNA replication and gene transcription.Once translocated to the extracellular space, they act as DAMPs that mediate in ammatory response and organ injury via PRRs typi ed by TLRs 9,17,18 .Previous articles showed that in patients with ARDS, circulating histones and histone proteins in the bronchoalveolar lavage uids were increased and contributed to the pathogenesis of ARDS 19 .Histone proteins induce endothelial or epithelial cell injury in the lungs and kidneys, which results in pulmonary edema and multiple organ failure with neutrophil in ltration in capillaries 17,20,21 .Furthermore, histones induce coagulopathy and thrombosis via active thrombin production by reducing thrombomodulin-dependent protein C activation 22 and recruiting brinogen to induce platelet aggregation 23 .In COVID-19, histone-mediated lung injury and thrombosis related to neutrophil extracellular traps (NETs) are also expected to be involved in the pathogenesis 24,25 .Our observations reinforce the importance of histones in the COVID-19 deterioration and the pathogenesis.
Extracellular histones originate from dead cells, and NETs of activated neutrophil origin as chromatin 18 or are actively released in soluble or vesicle-associated form by immune cells such as lymphocyte and monocyte/macrophage without cell death 26 .Furthermore, histone modulates proin ammatory responses on the cell surface in lymphocytes and monocyte/macrophages by translocating them from the nucleus, which is also unrelated to cell death 27 .Here, we show that in PBMCs collected from deteriorated patients, at the early stage of the disease before deterioration, HIST1 cluster gene expression is upregulated without a cell death signature.Therefore, it highlights the importance of histone expression and secretion or translocation to the membrane in PBMCs, other than NETs-mediated histone released, in the pathogenesis and detection of COVID-19 exacerbation at the early stages of the disease.
Our work further demonstrates that PIAS4 can be an upstream mediator associated with COVID-19 deterioration.PISA4 is a transcriptional corepressor that physically interacts with transcription factors such as TP53, STAT1, and NFκB [14][15][16] .Our in silico analysis predicts PIAS4 as an upstream transcription factor strongly enriched by HIST1 cluster genes that are upregulated, indicating that gene expression may be derepressed.Its function can explain this as a SUMO E3 ligase or redundant interaction network that modulates functions and subcellular localization of many proteins and regulates numerous signaling pathways, including in ammatory pathways such as NF-kB signaling in a context-dependent manner 13,14 .Intriguingly, PIAS4 is also physically interacts with viral proteins or DNA sites upon the infection, which regulates viral replication 28,29 .These functions may inhibit PIAS4 as an intranuclear transcriptional corepressor, resulting in the upregulation of HIST1 cluster gene expression.Thus, our conclusion is still hypothetical but can propose candidate molecules, a new mechanistic insight into the pathogenesis, and a marker for the disease progression of COVID-19.This study is not without limitations.PBMCs includes several subpopulations such as monocytes, lymphocytes and dendric cells, but the speci c cell types that exhibit HIST1 cluster gene upregulation are not determined.Additionally, the distribution of upregulated histone protein, e.g., cellular surface or circulation, remains to be uncovered.Future studies will validate the function of histone proteins and the expression and function of upstream regulators.
In conclusion, we have reported that HIST1 cluster gene expression is upregulated in PBMCs from patients with deteriorating COVID-19 at the early stage of the disease.Our results propose a molecular signature and mechanisms that involve PIAS4-mediated transcriptional regulation and in ammatory response.Histone-mediated lung injury could be linked to the pathogenesis and the deterioration of COVID-19.

Study participants
Patients with mild-to-moderate COVID-19 admitted to our COVID-19 subunit at Nishi-Kobe Medical Center within six days after the disease onset were eligible for inclusion between August 2020 and February 2021.SARS-CoV-2 infection was con rmed by RT-qPCR analysis.Also, blood for RNA-seq analysis was collected at admission.Disease severity was evaluated using the WHO de nition (meet any of the followings: oxygen saturation < 90%, respiratory rate > 30 breaths/min, and signs of severe respiratory distress or not).Based on the subsequent clinical courses, patients were grouped into two groups, deteriorated (developed severe diseases) and nondeteriorated (had only mild-to-moderate diseases).The baseline characteristics of patients with COVID-19, which include age, sex, the day of disease onset and sample collection, body temperature, pulse rate, SpO 2 , white blood cells, neutrophils, lymphocytes, monocytes, CRP, LDH, and D-dimer were extracted from medical records.This study was conducted following the World Medical Association's Declaration of Helsinki and approved by the ethical review board of Nishi-Kobe Medical Center (#2021-22).Informed consent was obtained from all study participants.

PBMCs isolation and total RNA extraction
Density gradient centrifugation was used to prepare PBMCs.In detail, Lymphoprep (STEMCELL TECHNOLOGIES, Canada) was added to blood samples and poured into SepMate(STEMCELL TECHNOLOGIES), followed by dilution using an equal volume of saline.After centrifugation at 1,200 g for 10 min, the layer containing PBMCs was transferred into a new tube and washed with saline.Then, the pellet was collected by centrifugation at 300 g for 8 min.Total RNA was extracted using Maxwell RSC instrument and Maxwell RSC simplyRNA Blood Kit (Promega, WI, USA) according to the manufacturer's instructions and stored at −80 °C for future analysis.

RNA-seq analysis
Total RNA from six deteriorated and six nondeteriorated patients with COVID-19 were analyzed.Libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) and subjected to sequencing with an Illumina Novaseq 6000 instrument (Illumina).Reads were trimmed using FASTQ groomer 30 and aligned to the GENCODE human reference genome GRCh37 (hg19, GENCODE website) using the HISAT2 function 31 on the Galaxy platform 32 .Gene transcripts were quanti ed using DESeq2 33 , and the volcano plot was generated by ggplot2 on Galaxy.Heatmap was generated by ClustVis 34