Background: 

Mycobacterium tuberculosis (Mtb) causes active tuberculosis (TB) in approximately 10 million people annually, resulting in 1.6 million deaths. TB and helminthiasis have a significant geographical overlap, and helminth infections can trigger immunological responses that dampen Th1 immunity, which is essential for Mtb containment. While B cells are known to modulate T cell responses, their role during helminth/TB co-infection remains unclear.

Objective: 

This study aimed to analyze the effect of helminth exposure on T cell activation in a T and B cell co-culture system.

Methods: 

T and B cells were isolated from healthy donor blood, stimulated with aCD3/aCD28, and exposed to Ascaris lumbricoides protein antigens (ASC) or Schistosoma mansoni soluble egg antigen (SM).

Results: 

B cells reduced T cell proliferation, and SM exposure partly attenuated this inhibitory effect on CD4 T cells. ASC exposure did not affect T cell proliferation but increased soluble IL-15Rα and surface IL-15Rα and IL-2/15Rβ on CD4 T cells. Restimulation with recombinant human IL-15 revealed reduced and unsustained STAT5 activation in CD4 and CD8 T cells in ASC-exposed co-cultures. Additional analysis showed altered phosphorylation of STAT1, STAT3, and STAT6, indicating broader impairment of IL-15 responsiveness and a dampened Th1 activation profile. BCL6/BLIMP-1 transcription factor expression of B cells in ASC-exposed co-cultures suggested a shift toward a more differentiated phenotype, such as plasma cell, memory B cells, or marginal zone MZ B cells, which are all typically BCL6low. The in vivo analysis of peripheral blood mononuclear cells from individuals in a helminth/TB endemic region showed increased frequencies of CD38hiCD24hi B regulatory cells in helminth infected, and MZ B cells (IgMhigh unswitched B cells) in both helminth-infected and helminth/TB co-infected individuals.

Conclusion: 

ASC antigen exposure modulates T cell activation through dynamic regulation of the IL-15-pathway and STAT signaling. These findings suggest that ASC exposure may help prevent prolonged IL-15-driven responses, potentially limiting excessive inflammation during TB. B cells in ASC-exposed co-cultures exhibit transcriptional changes consistent with a shift away from the germinal center phenotype toward more differentiated states.

In systemic lupus erythematosus (SLE) and other autoimmune diseases, B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play critical roles through three receptors, i.e., BAFF-Receptor (BAFF-R), transmembrane activator and calcium-modulator and cyclophilin ligand (CAML) interactor (TACI), and B-cell maturation antigen (BCMA), promoting the survival of self-reactive B cells and supporting their differentiation into antibody-producing cells. Several biologic therapies with anti-BAFF and anti-BAFF/APRIL agents induce profound suppression of immunoglobulin production and B-cell survival with variable safety profiles. While belimumab has demonstrated significant improvement of overall disease activity in clinical trials, some cases of de novo lupus nephritis have been reported suggesting a potential lack of protection in certain cases. The sustained reduction in B cell populations expressing regulatory markers, alongside a rapid decline in IL-10 levels following belimumab initiation, suggests that BAFF may have a previously underappreciated role in supporting the development or function of regulatory B cells (Bregs). Given the complex receptor interactions of BAFF and APRIL, as well as the heterogeneous phenotype of regulatory B cells and their different mechanisms for regulating the immune response, the involvement of BAFF in regulatory immune responses remains difficult to fully elucidate. This review explores the current evidence on the anti-BAFF/APRIL therapies and examines the unresolved question of whether BAFF exerts a context-dependent role in supporting regulatory B cell function.

Regulatory B (Breg) cells can dampen inflammation, autoreactivity, and transplant rejection. We investigated the frequencies, phenotypes, and function of Breg cells in granulomatosis with polyangiitis (GPA) to gain further knowledge as to whether there are numerical alterations or limitations of their ability to regulate T-cell function. Frequencies and phenotypes of CD24hiCD27+ and CD24hiCD38hi B-cells in the blood were determined with flow cytometry in 37 GPA patients (22 in remission and 15 with active disease) and 31 healthy controls (HC). A co-culture model was used to study the capacity of Breg cells to regulate T-cell activation and proliferation in cells from 10 GPA patients in remission and 12 HC. T-cell cytokine production in vitro and levels in plasma were determined with enzyme-linked immunosorbent assay. Frequencies of CD24hiCD27+ B-cells were reduced both during active disease and remission compared with HC (P = 0.005 and P = 0.010, respectively), whereas CD24hiCD38hi B-cells did not differ. Patient CD24hiCD27+ B-cells exhibited decreased expression of CD25 but increased expression of PD-L1 and PD-L2 during remission. B-cells from GPA patients regulated T-cell proliferation but failed to regulate interferon (IFN)-γproduction (median T-cells alone 222 ng/ml vs. T-cells + B-cells 207 ng/ml, P = 0.426). IFN-γwas also elevated in patient plasma samples (P = 0.016). In conclusion, GPA patients exhibit altered numbers and phenotypes of CD24hiCD27+ B-cells. This is accompanied by a disability to control T-cell production of Th1-type cytokines during remission, which might be of fundamental importance for the granulomatous inflammation that characterizes the chronic phase of this disease. © 2023 The Author(s). Published by Oxford University Press on behalf of the British Society for Immunology.

Neutrophil extracellular traps (NETs) and mitochondrial DNA (mtDNA) are inflammatory mediators involved in the development of type 1 diabetes (T1D). Pancreas-infiltrating neutrophils can release NETs, contributing to the inflammatory process. Levels of NETs are increased in serum from patients with T1D and mtDNA is increased in adult T1D patients. Our aim was to investigate extracellular DNA (NETs, mtDNA and nuclear DNA) in children with newly diagnosed T1D and in children at high risk of the disease. We also elucidated if extracellular DNA short after diagnosis could predict loss of endogenous insulin production. Samples were analysed for mtDNA and nuclear DNA using droplet digital PCR and NETs were assessed by a NET-remnants ELISA. In addition, in vitro assays for induction and degradation of NETs, as well as analyses of neutrophil elastase, HLA genotypes, levels of c-peptide, IL-1beta, IFN and autoantibodies (GADA, IA-2A, IAA and ZnT8A) were performed. In serum from children 10 days after T1D onset there was an increase in NETs (p=0.007), mtDNA (p<0.001) and nuclear DNA (p<0.001) compared to healthy children. The elevated levels were found only in younger children. In addition, mtDNA increased in consecutive samples short after onset (p=0.017). However, levels of extracellular DNA short after onset did not reflect future loss of endogenous insulin production. T1D serum induced NETs in vitro and did not deviate in the ability to degrade NETs. HLA genotypes and autoantibodies, except for ZnT8A, were not associated with extracellular DNA in T1D children. Serum from children with high risk of T1D showed fluctuating levels of extracellular DNA, sometimes increased compared to healthy children. Therefore, extracellular DNA in serum from autoantibody positive high-risk children does not seem to be a suitable biomarker candidate for prediction of T1D. In conclusion, we found increased levels of extracellular DNA in children with newly diagnosed T1D, which might be explained by an ongoing systemic inflammation.

Neutrophils operate as part of the innate defence in the skin and may eliminate the Borrelia spirochaete via phagocytosis, oxidative bursts, and hydrolytic enzymes. However, their importance in Lyme neuroborreliosis (LNB) is unclear. Neutrophil extracellular trap (NET) formation, which is associated with the production of reactive oxygen species, involves the extrusion of the neutrophil DNA to form traps that incapacitate bacteria and immobilise viruses. Meanwhile, NET formation has recently been studied in pneumococcal meningitis, the role of NETs in other central nervous system (CNS) infections has previously not been studied. Here, cerebrospinal fluid (CSF) samples from clinically well-characterised children (N = 111) and adults (N = 64) with LNB and other CNS infections were analysed for NETs (DNA/myeloperoxidase complexes) and elastase activity. NETs were detected more frequently in the children than the adults (p = 0.01). NET presence was associated with higher CSF levels of CXCL1 (p < 0.001), CXCL6 (p = 0.007), CXCL8 (p = 0.003), CXCL10 (p < 0.001), MMP-9 (p = 0.002), TNF (p = 0.02), IL-6 (p < 0.001), and IL-17A (p = 0.03). NETs were associated with fever (p = 0.002) and correlated with polynuclear pleocytosis (rs = 0.53, p < 0.0001). We show that neutrophil activation and active NET formation occur in the CSF samples of children and adults with CNS infections, mainly caused by Borrelia and neurotropic viruses. The role of NETs in the early phase of viral/bacterial CNS infections warrants further investigation.

How do you wash cells? Three out of four of our colleagues use experimental procedures during everyday lab-bench work that can severely impair data interpretation depending on how cells are handled. We show here that a subpopulation (2–3%) of human leukocytes immediately induce a yet unclassified lytic cell death, concomitant with discharge of chromatin entities and cell elimination, when placed in protein-free solutions (i.e., PBS and HBSS). DNA release was not restricted to hematopoietic cells but occurred also in HEK293T cells. Albumin, fetal bovine serum, polyethylene glycol, and Pluronic F-68 supplements prevented chromatin discharge. Expelled chromatin was devoid of surrounding membranes but maintained its original nuclear shape, although ∼10 times enlarged. These structures differed from DNA appearance after osmotic or detergent-induced cell lysis. Besides sounding a cautionary note to the neutrophil extracellular trap (NET) research community, in which ∼50% of all published studies used protein-free media for NET-formation, our study also provides a rapid tool for analysis of chromatin organization.

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

Background: Imatinib mesylate (Glivec, formerly STI-571) is a selective tyrosine kinase inhibitor used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, there are reports suggesting that imatinib could be atheroprotective by lowering plasma low-density lipoprotein (LDL). Aim: To investigate the potential inhibitory effect of imatinib on cholesterol uptake in human macrophages as well as its effect on matrix metalloproteinase (MMP) activity. Methods and results: Uptake of fluorescence-labeled LDL was analyzed using flow cytometry. Macrophages treated with imatinib showed a 23.5%, 27%, and 15% decrease in uptake of native LDL (p &lt; 0.05), acetylated LDL (p &lt; 0.01), and copper-modified oxidized LDL (p &lt; 0.01), respectively. Gel based zymography showed that secretion and activity of MMP-2 and MMP-9 were inhibited by imatinib. Using GeneChip Whole Transcript Expression array analysis, no obvious gene candidates involved in the mechanisms of cholesterol metabolism or MMP regulation were found to be affected by imatinib. Instead, we found that imatinib up-regulated microRNA 155 (miR155) by 43.8% and down-regulated ADAM metallopeptidase domain 28 (ADAM28) by 41.4%. Both genes could potentially play an atheroprotective role and would be interesting targets in future studies. Conclusion: Our results indicate that imatinib causes post-translational inhibition with respect to cholesterol uptake and regulation of MMP-2 and MMP-9. More research is needed to further evaluate the role of imatinib in the regulation of other genes and processes. (c) 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3-and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3-and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced clearance of NETs is important in AAV. However, not all ANCAs are pathogenic and some might possibly also aid in the clearance of NETs. A dual role for ANCAs in relation to circulating NET levels has been proposed because a negative correlation was observed between PR3-ANCA and NET remnants in patients in remission.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitute a group of vasculitides characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of ANCA in the circulation. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. NETs were first described to be involved in capture and elimination of pathogens but dysregulated production and/or clearance of NETs are thought to contribute to vessel inflammation in AAV; directly by damaging endothelial cells and indirectly by acting as a link between the innate and adaptive immune system through the generation of pathogenic PR3-ANCA and MPO-ANCA that can activate neutrophils. ANCA can, however, be found in all individuals and are therefore suggested to belong to the repertoire of natural antibodies produced by innate-like B cells, implying that not all ANCA are pathogenic. 

In paper I, we found neutrophils in patients to be more prone to undergo NETosis/necrosis spontaneously compared with neutrophils in healthy controls (HC), as well as that active patients possessed elevated levels of NETs in the circulation. Our results also suggest that ANCA during remission could contribute to the clearance of NETs as we observed an inverse relation between ANCA and NETs. In paper II, we observed neutrophils in patients to be more easily activated upon ANCA stimulation as they produced more ROS than neutrophils in HC. In paper III, we showed for the first time that cells of adaptive immunity (B and T cells) in addition to cells of innate immunity can release ET-like structures, in this case consisting of mitochondrial (mt) DNA. mtDNA can act as a damage-associated pattern molecule (DAMP) and promote inflammation, and increased levels of mtDNA has been observed in AAV. Our finding broadens our perspective of the possible roles of T and B cells in immunological responses, and should be further investigated in AAV. In paper IV, we observed reduced frequencies of MZ-like B cells, considered to be innate-like B cells that produce natural antibodies, and of the proposed regulatory B (Breg) cell populations CD24highCD27+ and CD25+CD27+ B cells in patients, particularly in those with active disease. We also observed the phenotypes of these different Breg cell populations to be different from the corresponding cells in HC.

We hypothesize that the increased activation potential by neutrophils in AAV to produce ROS and undergo NETosis/necrosis contribute to the excessive inflammation as well as an increased antigen load of PR3 and MPO, and that this in combination with dysregulation of innate-like B cells and Breg cells could lead to break of tolerance to these antigens and production of pathogenic autoantibodies. ANCA can in turn activate neutrophils to release NETs, suggesting a vicious circle in disease development.