Extensive acute and sustained changes to neutrophil proteomes post-SARS-CoV-2 infection.

BACKGROUND: Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery. METHODS: Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale. RESULTS: Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors. CONCLUSIONS: SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines.

Quantitative Analyses Reveal How Hypoxia Reconfigures the Proteome of Primary Cytotoxic T Lymphocytes.

Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how the proteome of primary murine CD8+ cytotoxic T lymphocytes (CTLs) is reconfigured in response to hypoxia in vitro. We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. Moreover, we determined that hypoxia could inhibit IL-2-induced proliferation cues and antigen-induced pro-inflammatory cytokine production in CTLs. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8+ T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts.

Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs.

Interleukin-2 (IL-2) and Janus kinases (JAKs) regulate transcriptional programs and protein synthesis to promote the differentiation of effector CD8+ cytotoxic T lymphocytes (CTLs). Using high-resolution mass spectrometry, we generated an in-depth characterization of how IL-2 and JAKs configure the CTL proteome to control CTL function. We found that IL-2 signaling through JAK1 and JAK3 (JAK1/3) increased the abundance of a key subset of proteins to induce the accumulation of critical cytokines and effector molecules in T cells. Moreover, IL-2 maintained the concentration of proteins that support core metabolic processes essential for cellular fitness. One fundamental insight was the dominant role for IL-2 in stimulating effector T cells to detect microenvironmental cues. IL-2-JAK1/3 signaling pathways thus increased the abundance of nutrient transporters, nutrient sensors, and critical oxygen-sensing molecules. These data provide key insights into how IL-2 promotes T cell function and highlight signaling mechanisms and transcription factors that integrate oxygen sensing to transcriptional control of CD8+ T cell differentiation.

Electrical slow waves in the mouse oviduct are dependent on extracellular and intracellular calcium sources.

Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca(2+) imaging were performed to examine the role of extracellular and intracellular Ca(2+) sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca(2+) channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca(2+) concentration ([Ca(2+)](o)) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca(2+) waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca(2+)](o). Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K(+) concentration ([K(+)](o)). Ni(2+) also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca(2+) waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps.