Recruitment of the lipid kinase Mss4 to the meiotic spindle pole promotes prospore membrane formation in Saccharomyces cerevisiae.

Spore formation in the budding yeast, Saccharomyces cerevisiae, involves de novo creation of four prospore membranes, each of which surrounds a haploid nucleus resulting from meiosis. The meiotic outer plaque (MOP) is a meiosis-specific protein complex associated with each meiosis II spindle pole body (SPB). Vesicle fusion on the MOP surface creates an initial prospore membrane anchored to the SPB. Ady4 is a meiosis-specific MOP component that stabilizes the MOP-prospore membrane interaction. We show that Ady4 recruits the lipid kinase, Mss4, to the MOP. MSS4 overexpression suppresses the ady4∆ spore formation defect, suggesting that a specific lipid environment provided by Mss4 promotes maintenance of prospore membrane attachment to MOPs. The meiosis-specific Spo21 protein is an essential structural MOP component. We show that the Spo21 N terminus contains an amphipathic helix that binds to prospore membranes. A mutant in SPO21 that removes positive charges from this helix shares phenotypic similarities to ady4∆. We propose that Mss4 generates negatively charged lipids in prospore membranes that enhance binding by the positively charged N terminus of Spo21, thereby providing a mechanism by which the MOP-prospore membrane interaction is stabilized.

Modulation of tissue resident memory T cells by glucocorticoids after acute cellular rejection in lung transplantation.

Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single-cell RNA and TCR sequencing on recipient-derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with rejection and compare them with T cells obtained from the same patients after treatment of rejection with high-dose systemic glucocorticoids. At the time of rejection, we found an oligoclonal expansion of cytotoxic CD8+ T cells that all persisted as tissue resident memory T cells after successful treatment. Persisting CD8+ allograft-resident T cells have reduced gene expression for cytotoxic mediators after therapy with glucocorticoids but accumulate around airways. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in situ expansion. We thus highlight the accumulation of cytotoxic, recipient-derived tissue resident memory T cells within the lung allograft that persist despite the administration of high-dose systemic glucocorticoids. The long-term clinical consequences of this persistence have yet to be characterized.

ß-Agonist exposure preferentially impacts lung macrophage cyclic AMP-related gene expression in asthma and asthma COPD overlap syndrome.

Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, bulk RNA sequencing from cultured macrophage (THP-1) cells after acute or prolonged ß-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort. THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute ß-agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations (P < 3.9e-111). Natural killer (NK) cells and type II alveolar epithelial cells displayed less robust network suppression (P < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiological parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.

Increased Alternative Complement Pathway Function and Improved Survival during Critical Illness.

Rationale: Complement is crucial for host defense but may also drive dysregulated inflammation. There is limited understanding of alternative complement function, which can amplify all complement activity, during critical illness.Objectives: We examined the function and key components of the alternative complement pathway in a series of critically ill patients and in a mouse pneumonia model.Methods: Total classical (CH50) and alternative complement (AH50) function were quantified in serum from 321 prospectively enrolled critically ill patients and compared with clinical outcomes. Alternative pathway (AP) regulatory factors were quantified by ELISA (n = 181) and examined via transcriptomics data from external cohorts. Wild-type, Cfb-/-, and C3-/- mice were infected intratracheally with Klebsiella pneumoniae (KP) and assessed for extrapulmonary dissemination.Measurements and Main Results: AH50 greater than or equal to median, but not CH50 greater than or equal to median, was associated with decreased 30-day mortality (adjusted odds ratio [OR], 0.53 [95% confidence interval (CI), 0.31-0.91]), independent of chronic liver disease. One-year survival was improved in patients with AH50 greater than or equal to median (adjusted hazard ratio = 0.59 [95% CI, 0.41-0.87]). Patients with elevated AH50 had increased levels of AP factors B, H, and properdin, and fewer showed a "hyperinflammatory" subphenotype (OR, 0.30 [95% CI, 0.18-0.49]). Increased expression of proximal AP genes was associated with improved survival in two external cohorts. AH50 greater than or equal to median was associated with fewer bloodstream infections (OR, 0.67 [95% CI, 0.45-0.98). Conversely, depletion of AP factors, or AH50 less than median, impaired in vitro serum control of KP that was restored by adding healthy serum. Cfb-/- mice demonstrated increased extrapulmonary dissemination and serum inflammatory markers after intratracheal KP infection compared with wild type.Conclusions: Elevated AP function is associated with improved survival during critical illness, possibly because of enhanced immune capacity.