Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

Cytokinopathy with aberrant cytotoxic lymphocytes and profibrotic myeloid response in SARS-CoV-2 mRNA vaccine-associated myocarditis.

Rare immune-mediated cardiac tissue inflammation can occur after vaccination, including after SARS-CoV-2 mRNA vaccines. However, the underlying immune cellular and molecular mechanisms driving this pathology remain poorly understood. Here, we investigated a cohort of patients who developed myocarditis and/or pericarditis with elevated troponin, B-type natriuretic peptide, and C-reactive protein levels as well as cardiac imaging abnormalities shortly after SARS-CoV-2 mRNA vaccination. Contrary to early hypotheses, patients did not demonstrate features of hypersensitivity myocarditis, nor did they have exaggerated SARS-CoV-2-specific or neutralizing antibody responses consistent with a hyperimmune humoral mechanism. We additionally found no evidence of cardiac-targeted autoantibodies. Instead, unbiased systematic immune serum profiling revealed elevations in circulating interleukins (IL-1ß, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix metalloproteases (MMP1, MMP8, MMP9, and TIMP1). Subsequent deep immune profiling using single-cell RNA and repertoire sequencing of peripheral blood mononuclear cells during acute disease revealed expansion of activated CXCR3+ cytotoxic T cells and NK cells, both phenotypically resembling cytokine-driven killer cells. In addition, patients displayed signatures of inflammatory and profibrotic CCR2+ CD163+ monocytes, coupled with elevated serum-soluble CD163, that may be linked to the late gadolinium enhancement on cardiac MRI, which can persist for months after vaccination. Together, our results demonstrate up-regulation in inflammatory cytokines and corresponding lymphocytes with tissue-damaging capabilities, suggesting a cytokine-dependent pathology, which may further be accompanied by myeloid cell-associated cardiac fibrosis. These findings likely rule out some previously proposed mechanisms of mRNA vaccine--associated myopericarditis and point to new ones with relevance to vaccine development and clinical care.

Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines.

BACKGROUND: COVID-19 manifests with a wide spectrum of clinical phenotypes, ranging from asymptomatic and mild to severe and critical. Severe and critical COVID-19 patients are characterized by marked changes in the myeloid compartment, especially monocytes. However, little is known about the epigenetic alterations that occur in these cells during hyperinflammatory responses in severe COVID-19 patients. METHODS: In this study, we obtained the DNA methylome and transcriptome of peripheral blood monocytes from severe COVID-19 patients. DNA samples extracted from CD14 + CD15- monocytes of 48 severe COVID-19 patients and 11 healthy controls were hybridized on MethylationEPIC BeadChip arrays. In parallel, single-cell transcriptomics of 10 severe COVID-19 patients were generated. CellPhoneDB was used to infer changes in the crosstalk between monocytes and other immune cell types. RESULTS: We observed DNA methylation changes in CpG sites associated with interferon-related genes and genes associated with antigen presentation, concordant with gene expression changes. These changes significantly overlapped with those occurring in bacterial sepsis, although specific DNA methylation alterations in genes specific to viral infection were also identified. We also found these alterations to comprise some of the DNA methylation changes occurring during myeloid differentiation and under the influence of inflammatory cytokines. A progression of DNA methylation alterations in relation to the Sequential Organ Failure Assessment (SOFA) score was found to be related to interferon-related genes and T-helper 1 cell cytokine production. CellPhoneDB analysis of the single-cell transcriptomes of other immune cell types suggested the existence of altered crosstalk between monocytes and other cell types like NK cells and regulatory T cells. CONCLUSION: Our findings show the occurrence of an epigenetic and transcriptional reprogramming of peripheral blood monocytes, which could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these patients.

Maximizing insights from monogenic immune disorders.

Monogenic immune disorders provide unprecedented insights into the consequences of disrupting single genes in humans, thereby informing our understanding of fundamental immune function and disease. Genomics has accelerated monogenic disease discovery while also revealing the complexity of human disease, where several factors beyond the genome can govern pathogenesis. At this juncture, the optimal path forward will focus on maximizing basic and translational immunology insights from these disorders. This pursuit will be most direct and impactful if human disease gene discovery is paired with mechanistic studies employing integrative omics and mouse modeling to leverage their unique strengths.

Quantifying Sample Collection and Processing Impacts on Fiber-Based Tear Fluid Chemical Analysis.

Purpose: Noninvasive analyses of tear fluid from humans and animal models in clinical and research settings most commonly use absorbent material for collection and processing. Still, the impact of these analytical techniques on tear chemical analyses remains largely unknown. The purpose of this study was to quantify the impacts of phenol red thread fiber-based tear sample collection and processing on the primary amine content. Methods: Human tears were collected by placing the folded end of phenol red thread on the palpebral conjunctiva of the right eye for 20 seconds. The wetted thread was then processed using elution or extraction, and capillary electrophoresis with light-emitting diode-induced fluorescence detection was used for analysis and quantitation. Results: Distinct processing methods impacted tear analysis differently. Primary amines adsorbed onto the thread partitioned in a chromatographic manner and thus any single portion of the wetted thread might not be representative of the whole sample. Quantitative assessment of five small molecule standards after on-thread processing showed significant overestimation of the actual concentration, with increased accuracy for larger volume samples. Yet collection of larger tear volumes introduced error in volume determination owing to evaporation and reduced small molecule separation resolution. Conclusions: These results indicated that absorption-based tear fluid collection and processing significantly alter chemical content analysis, suggesting that the impacts of methods used should be regularly evaluated to standardize results drawn from different studies. Translational Relevance: This study identifies potential inconsistencies and inaccuracies in tear analyses that are widespread across the published literature and clinical care.

Thread-based assay for quantitative small molecule analysis of mice tear fluid by capillary electrophoresis.

Tear fluid plays blood-like roles in the cornea, and changes in its chemical composition may be reflective of ocular surface disease pathogenesis. Studies of mice tears are limited by the small volume available for collection and difficulty in obtaining representative samples. Here, we establish a non-invasive assay for small volume analysis of small molecules in mice tears that requires no pre-treatment of mice. To the best of our knowledge, this is the first small molecule analysis of mice tears. Nanoliters of mice tears (70 ± 25 nL) was collected via a single insertion of phenol red thread in the corner of the eye without anesthesia to prevent any tear production alteration. The processing and elution of tear samples were optimized for minimal sample handling and dilution while maintaining high separation resolution. A capillary electrophoresis separation with light-emitting diode-induced fluorescence detection was developed for the analysis of primary amine-containing small molecules. The levels of arginine, alanine, aspartate, and glutamate after elution were in the micromolar range as seen in human tears. However, taurine and histamine levels were decreased and increased, respectively, compared to human tears, which may be indicative of restraint-induced emotional stress. No significant differences were seen for any of the small molecules between 20-week-old ND4 Swiss Webster females and 12-week-old CD-1 males (N = 3). The developed assay represents a means to assess the chemical composition of tear fluid in mouse models of human disease, which could significantly improve our understanding of ocular surface diseases. Graphical abstract ᅟ.