Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies.

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that ∼50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (∼8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual's total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

Exposure to extracellular vesicles from Pseudomonas aeruginosa result in loss of DNA methylation at enhancer and DNase hypersensitive site regions in lung macrophages.

Various pathogens use differing strategies to evade host immune response including modulating the host's epigenome. Here, we investigate if EVs secreted from P. aeruginosa alter DNA methylation in human lung macrophages, thereby potentially contributing to a dysfunctional innate immune response. Using a genome-wide DNA methylation approach, we demonstrate that P. aeruginosa EVs alter certain host cell DNA methylation patterns. We identified 1,185 differentially methylated CpGs (FDR < 0.05), which were significantly enriched for distal DNA regulatory elements including enhancer regions and DNase hypersensitive sites. Notably, all but one of the 1,185 differentially methylated CpGs were hypomethylated in association with EV exposure. Significantly hypomethylated CpGs tracked to genes including AXL, CFB and CCL23. Gene expression analysis identified 310 genes exhibiting significantly altered expression 48 hours post P. aeruginosa EV treatment, with 75 different genes upregulated and 235 genes downregulated. Some CpGs associated with cytokines such as CSF3 displayed strong negative correlations between DNA methylation and gene expression. Our infection model illustrates how secreted products (EVs) from bacteria can alter DNA methylation of the host epigenome. Changes in DNA methylation in distal DNA regulatory regions in turn can modulate cellular gene expression and potential downstream cellular processes.

Extracellular Vesicles from Pseudomonas aeruginosa Suppress MHC-Related Molecules in Human Lung Macrophages.

Pseudomonas aeruginosa, a Gram-negative bacterium, is one of the most common pathogens colonizing the lungs of cystic fibrosis patients. P. aeruginosa secrete extracellular vesicles (EVs) that contain LPS and other virulence factors that modulate the host's innate immune response, leading to an increased local proinflammatory response and reduced pathogen clearance, resulting in chronic infection and ultimately poor patient outcomes. Lung macrophages are the first line of defense in the airway innate immune response to pathogens. Proper host response to bacterial infection requires communication between APC and T cells, ultimately leading to pathogen clearance. In this study, we investigate whether EVs secreted from P. aeruginosa alter MHC Ag expression in lung macrophages, thereby potentially contributing to decreased pathogen clearance. Primary lung macrophages from human subjects were collected via bronchoalveolar lavage and exposed to EVs isolated from P. aeruginosa in vitro. Gene expression was measured with the NanoString nCounter gene expression assay. DNA methylation was measured with the EPIC array platform to assess changes in methylation. P. aeruginosa EVs suppress the expression of 11 different MHC-associated molecules in lung macrophages. Additionally, we show reduced DNA methylation in a regulatory region of gene complement factor B (CFB) as the possible driving mechanism of widespread MHC gene suppression. Our results demonstrate MHC molecule downregulation by P. aeruginosa-derived EVs in lung macrophages, which is consistent with an immune evasion strategy employed by a prokaryote in a host-pathogen interaction, potentially leading to decreased pulmonary bacterial clearance.

DNA Methylation Changes in Regional Lung Macrophages Are Associated with Metabolic Differences.

A number of pulmonary diseases occur with upper lobe predominance, including cystic fibrosis and smoking-related chronic obstructive pulmonary disease. In the healthy lung, several physiologic and metabolic factors exhibit disparity when comparing the upper lobe of the lung to lower lobe, including differences in oxygenation, ventilation, lymphatic flow, pH, and blood flow. In this study, we asked whether these regional differences in the lung are associated with DNA methylation changes in lung macrophages that could potentially lead to altered cell responsiveness upon subsequent environmental challenge. All analyses were performed using primary lung macrophages collected via bronchoalveolar lavage from healthy human subjects with normal pulmonary function. Epigenome-wide DNA methylation was examined via Infinium MethylationEPIC (850K) array and validated by targeted next-generation bisulfite sequencing. We observed 95 CpG loci with significant differential methylation in lung macrophages, comparing upper lobe to lower lobe (all false discovery rate < 0.05). Several of these genes, including CLIP4, HSH2D, NR4A1, SNX10, and TYK2, have been implicated as participants in inflammatory/immune-related biological processes. Functionally, we identified phenotypic differences in oxygen use, comparing upper versus lower lung macrophages. Our results support a hypothesis that epigenetic changes, specifically DNA methylation, at a multitude of gene loci in lung macrophages are associated with metabolic differences regionally in lung.

Functional and metabolic impairment in cigarette smoke-exposed macrophages is tied to oxidative stress.

Cigarette smoke inhalation exposes the respiratory system to thousands of potentially toxic substances and causes chronic obstructive pulmonary disease (COPD). COPD is characterized by cycles of inflammation and infection with a dysregulated immune response contributing to disease progression. While smoking cessation can slow the damage in COPD, lung immunity remains impaired. Alveolar macrophages (AMΦ) are innate immune cells strategically poised at the interface between lungs, respiratory pathogens, and environmental toxins including cigarette smoke. We studied the effects of cigarette smoke on model THP-1 and peripheral blood monocyte derived macrophages, and discovered a marked inhibition of bacterial phagocytosis which was replicated in primary human AMΦ. Cigarette smoke decreased AMΦ cystic fibrosis transmembrane conductance regulator (CFTR) expression, previously shown to be integral to phagocytosis. In contrast to cystic fibrosis macrophages, smoke-exposed THP-1 and AMΦ failed to augment phagocytosis in the presence of CFTR modulators. Cigarette smoke also inhibited THP-1 and AMΦ mitochondrial respiration while inducing glycolysis and reactive oxygen species. These effects were mitigated by the free radical scavenger N-acetylcysteine, which also reverted phagocytosis to baseline levels. Collectively these results implicate metabolic dysfunction as a key factor in the toxicity of cigarette smoke to AMΦ, and illuminate avenues of potential intervention.

Genome-wide DNA methylation profiling shows a distinct epigenetic signature associated with lung macrophages in cystic fibrosis.

BACKGROUND: Lung macrophages are major participants in the pulmonary innate immune response. In the cystic fibrosis (CF) lung, the inability of lung macrophages to successfully regulate the exaggerated inflammatory response suggests dysfunctional innate immune cell function. In this study, we aim to gain insight into innate immune cell dysfunction in CF by investigating alterations in DNA methylation in bronchoalveolar lavage (BAL) cells, composed primarily of lung macrophages of CF subjects compared with healthy controls. All analyses were performed using primary alveolar macrophages from human subjects collected via bronchoalveolar lavage. Epigenome-wide DNA methylation was examined via Illumina MethylationEPIC (850 K) array. Targeted next-generation bisulfite sequencing was used to validate selected differentially methylated CpGs. Methylation-based sample classification was performed using the recursively partitioned mixture model (RPMM) and was tested against sample case-control status. Differentially methylated loci were identified by fitting linear models with adjustment of age, sex, estimated cell type proportions, and repeat measurement. RESULTS: RPMM class membership was significantly associated with the CF disease status (P = 0.026). One hundred nine CpG loci were differentially methylated in CF BAL cells (all FDR ≤ 0.1). The majority of differentially methylated loci in CF were hypo-methylated and found within non-promoter CpG islands as well as in putative enhancer regions and DNase hyper-sensitive regions. CONCLUSIONS: These results support a hypothesis that epigenetic changes, specifically DNA methylation at a multitude of gene loci in lung macrophages, may participate, at least in part, in driving dysfunctional innate immune cells in the CF lung.

Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling.

There are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for miRNA profiling studies. miRNA is an attractive candidate as a potential biomarker because of the high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. We present a comparison of small RNA recovery and stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of miRNA from cell-free urine and urine extracellular vesicles (EVs). Using an affinity slurry for isolation of small RNA from urine, we found that urine samples held at room temperature (20°C) for up to 8 hours before processing yield the highest amounts of intact small RNAs from EVs. Some miRNA is lost from urine samples when held 2°C to 4°C and/or frozen before EV isolation, likely because of EV entrapment in uromodulin precipitates. However, we found that a simple 5-minute incubation of urine containing cold-induced precipitate at 37°C resolubilizes much of this precipitate and results in an increased recovery of EVs and miRNAs. Finally, small RNA integrity can be compromised when whole urine is held at 37°C for as little as 4 hours and is not conducive to efficient miRNA profiling.