Tipping the Scale: Atheroprotective IgM-Producing B Cells in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease whose progression is fueled by proinflammatory moieties and limited by anti-inflammatory mediators. Whereas oxidative damage and the generation of oxidation-specific epitopes that act as damage-associated molecular patterns are highly inflammatory, IgM antibodies produced by B-1 and marginal zone B cells counteract unrestricted inflammation by neutralizing and encouraging clearance of these proinflammatory signals. In this review, we focus on describing the identities of IgM-producing B cells in both mice and humans, elaborating the mechanisms underlying IgM production, and discussing the potential strategies to augment the production of atheroprotective IgM. In addition, we will discuss promising therapeutic interventions in humans to help tip the scale toward augmentation of IgM production and to provide atheroprotection.

Single-cell profiling of CD11c+ B cells in atherosclerosis.

Circulating CD11c+ B cells, a novel subset of activated B cells, have been linked to autoimmunity and shown to expand with age. Atherosclerosis is an age-associated disease that involves innate and adaptive immune responses to modified self-antigens. Yet, the expression of CD11c on specific B-cell subtypes and its link to atherosclerosis are poorly understood. In this study, we characterized the frequency of CD11c+ B cells in tissues in mice with aging. We observed an age-associated increase in CD11c+ B cells in the spleen and bone marrow of ApoE-/- mice, and this was associated with an increase in aortic plaque. In addition, we also utilized single-cell multi-omics profiling of 60 human subjects undergoing advanced imaging for coronary artery disease (CAD) to subtype CD11c+ B cells and determine their frequency in subjects with high and low severity of CAD. Using unsupervised clustering, we identified four distinct clusters of CD11c+ B cells, which include CD27 and IgD double negative 2 (DN2), age-associated (ABC), CD11c+ unswitched memory (USWM), and activated Naïve (aNav) B cells. We observed an increase in the frequency of both ABC B cells and DN2 B cells in patients with high CAD severity. Pathway analysis further demonstrated augmentation of autophagy, IFNg signaling, and TLR signaling in DN2 cells in high-severity CAD patients. On the other hand, an increase in the negative regulator of BCR signaling through CD72 was found in ABC cells in low-severity CAD patients. Through investigating scRNAseq of atheroma, these DN2 cells were also found to infiltrate human coronary atheroma.

A multifaceted cellular damage repair and prevention pathway promotes high-level tolerance to ß-lactam antibiotics.

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level ß-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that ß-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to ß-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against ß-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients.

The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID19 Patients.

The development of neutralizing antibodies (nAb) against SARS-CoV-2, following infection or vaccination, is likely to be critical for the development of sufficient population immunity to drive cessation of the COVID19 pandemic. A large number of serologic tests, platforms and methodologies are being employed to determine seroprevalence in populations to select convalescent plasmas for therapeutic trials, and to guide policies about reopening. However, tests have substantial variability in sensitivity and specificity, and their ability to quantitatively predict levels of nAb is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma using commercially available SARS-CoV- 2 detection tests and in-house ELISA assays and correlated serological measurements with nAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have varying degrees of accuracy in predicting nAb activity. We found the Ortho Anti-SARS-CoV-2 Total Ig and IgG high throughput serological assays (HTSAs), as well as the Abbott SARS-CoV-2 IgG assay, quantify levels of antibodies that strongly correlate with nAb assays and are consistent with gold-standard ELISA assay results. These findings provide immediate clinical relevance to serology results that can be equated to nAb activity and could serve as a valuable 'roadmap' to guide the choice and interpretation of serological tests for SARS-CoV-2.

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation.

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by 'autolysins', enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low-salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.