Glucocorticoids (GC) are important in the regulation of selection and apoptosis of CD4⁺CD8⁺ double-positive (DP) thymocytes. The pronounced GC-sensitivity of DP thymocytes, observed earlier, might be due to the combination of classical (genomic) and alternative (non-genomic) glucocorticoid receptor (GR) signaling events modifying activation or apoptotic pathways. In particular, the previously demonstrated mitochondrial translocation of activated GR in DP thymocytes offered a fascinating explanation for their pronounced GC-induced apoptosis sensitivity. However, the fine molecular details how the mitochondrial translocation of GR might regulate apoptosis remained unclear. Therefore, in the present study, we intended to examine which apoptotic pathways could be involved in GC-induced thymocyte apoptosis. Furthermore we investigated the potential relationship between the GR and Bcl-2 proteins. Using an in vitro test system, thymocytes from 4-week-old BALB/c mice, were treated with the GC-analogue dexamethasone (DX). Bax accumulated in mitochondria upon DX treatment. Mitochondrial GR showed association with members of the Bcl-2 family: Bak, Bim, Bcl-x_L. Elevated Cytochrome C, and active caspase-3, -8, and -9 levels were detected in thymocytes after DX treatment. These results support the hypothesis that in early phases of GC-induced thymocyte apoptosis, the mitochondrial pathway plays a crucial role, confirmed by the release of Cytochrome C and the activation of caspase-9. The activation of caspase-8 was presumably due to cross-talk between apoptotic signaling pathways. We propose that the GC-induced mitochondrial accumulation of Bax and the interaction between the GR and Bim, Bcl-x_L and Bak could play a role in the regulation of thymocyte apoptosis.

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.

Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180⁺ cells was the highest in the CD19⁺CD27⁺IgD⁺ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.

Contradictory reports are available on vaccine-associated hyperstimulation of the immune system, provoking the formation of pathological autoantibodies. Despite being interconnected within the same network, the role of the quieter, yet important non-pathological and natural autoantibodies (nAAbs) is less defined. We hypothesize that upon a prompt immunological trigger, physiological nAAbs also exhibit a moderate plasticity. We investigated their inducibility through aged and recent antigenic triggers. Anti-viral antibodies (anti-MMR n = 1739 and anti-SARS-CoV-2 IgG n = 330) and nAAbs (anti-citrate synthase IgG, IgM n = 1739) were measured by in-house and commercial ELISAs using Croatian (Osijek) anonymous samples with documented vaccination backgrounds. The results were subsequently compared for statistical evaluation. Interestingly, the IgM isotype nAAb showed a statistically significant connection with anti-MMR IgG seropositivity (p < 0.001 in all cases), while IgG isotype nAAb levels were elevated in association with anti-SARS CoV-2 specific seropositivity (p = 0.019) and in heterogeneous vaccine regimen recipients (unvaccinated controls vector/mRNA vaccines p = 0.002). Increasing evidence supports the interplay between immune activation and the dynamic expansion of nAAbs. Consequently, further questions may emerge regarding the ability of nAAbs silently shaping the effectiveness of immunization. We suggest re-evaluating the impact of nAAbs on the complex functioning of the immunological network.

Despite the abundance of data on the COVID-19 vaccine-induced immune activation, the impact of natural autoantibodies (nAAbs) on these processes is less well defined. Therefore, we investigated potential connections between vaccine efficacy and nAAb levels. We were also interested in the impact of immunomodulatory therapies on vaccine efficacy. Clinical residual samples were used for the assessment of the COVID-19 vaccine-elicited immune response (IR) (n=255), as well as for the investigation of the immunization-associated expansion of the nAAb pool (n=185). In order to study the potential interaction between immunomodulatory therapies and the vaccine-induced IR, untreated, healthy individuals and patients receiving anti-TNFα or anti-IL-17 therapies were compared (n total =45). In-house ELISAs (anticitrate synthase, anti-HSP60 and-70) and commercial ELISAs (anti-SARS-CoV-2 ELISAs IgG, IgA, NeutraLISA and IFN-γ release assay ‘IGRA’) were applied. We found significant differences in the IR given to different vaccines. Moreover, nAAb levels showed plasticity in response to anti-COVID-19 immunization. We conclude that our findings may support the theorem about the non-specific beneficial ‘side effects’ of vaccination, including the broadening of the nAAb repertoire. Considering immunomodulation, we suggest that anti-TNFα and anti-IL17 treatments may interfere negatively with MALT-associated IR, manifested as decreased IgA titers; however, the modest sample numbers of the herein presented model might be a limiting factor of reaching a more comprehensive conclusion.

The function of natural autoantibodies (nAAbs) in maintaining immunological tolerance has been comprehensively explained; however, their function in pregnant patients dealing with autoimmune diseases has not been thoroughly investigated. As Hashimoto’s thyroiditis (HT) is the predominant organ-specific autoimmune condition of women of childbearing age, this study’s objective was to evaluate IgM and IgG nAAbs targeting mitochondrial citrate synthase (CS) and heat shock proteins (Hsp60 and Hsp70) in women diagnosed with HT who were pregnant (HTP). Serum samples collected from HTP and healthy pregnant (HP) women in the first and third trimesters were tested using in-house-developed enzyme-linked immunosorbent assays (ELISAs). Our findings indicate the stability of nAAbs against CS and Hsps throughout the pregnancies of both healthy women and those with HT. However, during both trimesters, HTP patients displayed elevated levels of IgM isotype nAAbs against Hsp60 and Hsp70 compared to HP women, suggesting a regulatory role of IgM nAAbs during the pregnancies of patients with HT. Nonetheless, levels of IgG isotype nAAbs against Hsps were lower solely in the third trimester among HTP patients, resulting in a higher IgM/IgG ratio, which indicates their importance in alterations of the nAAb network during pregnancy in patients with HT.