Overactivation of the complement system has been characterized in severe COVID-19 cases. Complement components are known to trigger NETosis via the coagulation cascade and have also been reported in human tracheobronchial epithelial cells. In this longitudinal study, we investigated systemic and local complement activation and NETosis in COVID-19 patients that underwent mechanical ventilation. Results confirmed significantly higher baseline levels of serum C5a (24.5 ± 39.0 ng/mL) and TCC (11.03 ± 8.52 µg/mL) in patients compared to healthy controls (p < 0.01 and p < 0.0001, respectively). Furthermore, systemic NETosis was significantly augmented in patients (5.87 (±3.71) × 10⁶ neutrophils/mL) compared to healthy controls (0.82 (±0.74) × 10⁶ neutrophils/mL) (p < 0.0001). In tracheal fluid, baseline TCC levels but not C5a and NETosis, were significantly higher in patients. Kinetic studies of systemic complement activation revealed markedly higher levels of TCC and CRP in nonsurvivors compared to survivors. In contrast, kinetic studies showed decreased local NETosis in tracheal fluid but comparable local complement activation in nonsurvivors compared to survivors. Systemic TCC and NETosis were significantly correlated with inflammation and coagulation markers. We propose that a ratio comprising systemic inflammation, complement activation, and chest X-ray score could be rendered as a predictive parameter of patient outcome in severe SARS-CoV-2 infections.

Antigen-experienced immune cells migrate back to the bone marrow (BM), where they are maintained in BM survival niches for an extended period. The composition of T cell subpopulations in the BM changes with age, leading to an accumulation of highly differentiated T cells and a loss of naïve T cells. While innate immune cells are also affected by age, little is known about interactions between different adaptive immune cell populations maintained in the BM. In this study, the phenotype and function of innate and adaptive immune cells isolated from human BM and peripheral blood (PB) was analysed in detail using flow cytometry, to determine if the accumulation of highly differentiated T and B cells, supported by the BM niches, limits the maintenance of other immune cells, or affects their functions such as providing protective antibody concentrations. Total T cells increase in the BM with age, as do highly differentiated CD8+ T cells which no longer express the co-stimulatory molecule CD28, while natural killer T (NKT) cells, monocytes, B cells, and naïve CD8+ T cells all decrease in the BM with age. A negative correlation of total T cells with B cells was observed in the BM. The percentage of B cells in the BM negatively correlated with highly differentiated CD8+CD28− T cells, replicative-senescent CD8+CD57+ T cells, as well as the CD8+CD28−CD57+ population. Similar correlations were seen between B cells and the frequency of highly differentiated T cells producing pro-inflammatory molecules in the BM. Interestingly, plasma concentrations of diphtheria-specific antibodies negatively correlated with highly differentiated CD8+CD57+ T cells as well as with exhausted central memory CD8+ and CD4+ T cells in the BM. A negative impact on diphtheria-specific antibodies was also observed for CD8+ T cells expressing senescence associated genes such as the cell cycle regulator p21 (CDKN1A), KLRG-1, and elevated levels of reactive oxygen species (ROS). Our data suggest that the accumulation and maintenance of highly differentiated, senescent, and exhausted T cells in the BM, particularly in old age, may interfere with the survival of other cell populations resident in the BM such as monocytes and B cells, leading to reduced peripheral diphtheria antibody concentrations as a result. These findings further highlight the importance of the BM in the long-term maintenance of immunological memory.

Aging is characterized by reduced immune responses, a process known as immunosenescence. Shortly after their generation, antigen-experienced adaptive immune cells, such as CD8⁺ and CD4⁺ T cells, migrate into the bone marrow (BM), in which they can be maintained for long periods of time within survival niches. Interestingly, we recently observed how oxidative stress may negatively support the maintenance of immunological memory in the BM in old age. To assess whether the generation and maintenance of immunological memory could be improved by scavenging oxygen radicals, we vaccinated 18-months (old) and 3-weeks (young) mice with alum-OVA, in the presence/absence of antioxidants vitamin C (Vc) and/or N-acetylcysteine (NAC). To monitor the phenotype of the immune cell population, blood was withdrawn at several time-points, and BM and spleen were harvested 91 days after the first alum-OVA dose. Only in old mice, memory T cell commitment was boosted with some antioxidant treatments. In addition, oxidative stress and the expression of pro-inflammatory molecules decreased in old mice. Finally, changes in the phenotype of dendritic cells, important regulators of T cell activation, were additionally observed. Taken together, our data show that the generation and maintenance of memory T cells in old age may be improved by targeting oxidative stress.

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients’ blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients’ sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors.

Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis.