Age-related changes of the innate immune system of the palatine tonsil in a healthy cohort.

Although tonsillectomy is performed frequently, the role of palatine tonsils in life long immune protection or tolerance is still debated and the consequences of their removal for the immune system are of general interest. We analysed the tonsillar myeloid compartment in healthy subjects across a wide range of age (64% male; age range: 3 - 85 years) and compared its composition to the peripheral blood. We could observe a strong accumulation of all granulocyte subsets in the aging tonsil, which was most pronounced for basophils and mast cells. On functional level, an increase of CD163 and CD206 expression among monocytes and an increase of neutrophils expressing the inhibitory FcγRIIb correlated with increasing age. While the age-related shift of the leukocyte composition towards monocytes in blood is not reflected in tonsils, the increasing immunoregulatory phenotype of tonsilar monocytes is potentially counteracting the phenomenon of inflammaging at higher age.

Mediterranean diet component oleic acid increases protective lipid mediators and improves trabecular bone in a Porphyromonas gingivalis inoculation model.

AIM: Therapeutic modulation of bacterial-induced inflammatory host response is being investigated in gingival inflammation and periodontal disease pathology. Therefore, dietary intake of the monounsaturated fatty acid (FA) oleic acid (OA (C18:1)), which is the main component of Mediterranean-style diets, and saturated FA palmitic acid (PA (C16:0)), which is a component of Western-style diets, was investigated for their modifying potential in an oral inoculation model of Porphyromonas gingivalis. MATERIALS AND METHODS: Normal-weight C57BL/6-mice received OA- or PA-enriched diets (PA-ED, OA-ED, PA/OA-ED) or normal standard diet for 16 weeks and were inoculated with P. gingivalis/placebo (n = 12/group). Gingival inflammation, alveolar bone structure, circulating lipid mediators, and in vitro cellular response were determined. RESULTS: FA treatment of P. gingivalis-lipopolysaccharide-incubated gingival fibroblasts (GFbs) modified inflammatory activation, which only PA exacerbated with concomitant TNF-α stimulation. Mice exhibited no signs of acute inflammation in gingiva or serum and no inoculation- or nutrition-associated changes of the crestal alveolar bone. However, following P. gingivalis inoculation, OA-ED improved oral trabecular bone micro-architecture and enhanced circulating pro-resolving mediators resolvin D4 (RvD4) and 4-hydroxydocosahexaenoic acid (4-HDHA), whereas PA-ED did not. In vitro experiments demonstrated significantly improved differentiation in RvD4- and 4-HDHA-treated primary osteoblast cultures and reduced the expression of osteoclastogenic factors in GF. Further, P. gingivalis infection of OA-ED animals led to a serum composition that suppressed osteoclastic differentiation in vitro. CONCLUSIONS: Our results underline the preventive impact of Mediterranean-style OA-EDs by indicating their pro-resolving nature beyond anti-inflammatory properties.

Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity.

Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)-the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate "young-like" HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs.

Osteoarthritis-Induced Metabolic Alterations of Human Hip Chondrocytes.

Osteoarthritis (OA) alters chondrocyte metabolism and mitochondrial biology. We explored whether OA and non-OA chondrocytes show persistent differences in metabolism and mitochondrial function and different responsiveness to cytokines and cAMP modulators. Hip chondrocytes from patients with OA or femoral neck fracture (non-OA) were stimulated with IL-1ß, TNF, forskolin and opioid peptides. Mediators released from chondrocytes were measured, and mitochondrial functions and glycolysis were determined (Seahorse Analyzer). Unstimulated OA chondrocytes exhibited significantly higher release of IL-6, PGE2 and MMP1 and lower production of glycosaminoglycan than non-OA chondrocytes. Oxygen consumption rates (OCR) and mitochondrial ATP production were comparable in unstimulated non-OA and OA chondrocytes, although the non-mitochondrial OCR was higher in OA chondrocytes. Compared to OA chondrocytes, non-OA chondrocytes showed stronger responses to IL-1ß/TNF stimulation, consisting of a larger decrease in mitochondrial ATP production and larger increases in non-mitochondrial OCR and NO production. Enhancement of cAMP by forskolin prevented IL-1ß-induced mitochondrial dysfunction in OA chondrocytes but not in non-OA chondrocytes. Endogenous opioids, present in OA joints, influenced neither cytokine-induced mitochondrial dysfunction nor NO upregulation. Glycolysis was not different in non-OA and OA chondrocytes, independent of stimulation. OA induces persistent metabolic alterations, but the results suggest upregulation of cellular mechanisms protecting mitochondrial function in OA.

Persistent T-Cell Reactivity in a Seronegative Patient after SARS-CoV-2 Infection and One Vaccination.

We present here a 64-year-old male participant of the CoNAN study who experienced a PCR-confirmed mild SARS-CoV-2 infection but did not develop any measurable antibody response. Additionally, after vaccination with ChAdOx1 (AstraZeneca, Cambridge, UK) 11 months later, no antibodies were detected in six serological tests three weeks after the vaccination. When we assessed T-helper (Th) cell immunity, SARS-CoV-2-specific Th cells produced detectable amounts of IFNγ and TNF six weeks after the infection. A robust T-cell immunity remained detectable at least until six months after the infection and was boosted by the vaccination thereafter. This case report points out that an assessment of a prior infection or a vaccine response based solely on antibody detection might have limitations in individual patients.

Persistent humoral and CD4+ TH cell immunity after mild SARS-COV-2 infection-The CoNAN long-term study.

Understanding persistent cellular and humoral immune responses to SARS-CoV-2 will be of major importance to terminate the ongoing pandemic. Here, we assessed long-term immunity in individuals with mild COVID-19 up to 1 year after a localized SARS-CoV-2 outbreak. CoNAN was a longitudinal population-based cohort study performed 1.5 months, 6 months, and 12 months after a SARS-CoV-2 outbreak in a rural German community. We performed a time series of five different IgG immunoassays assessing SARS-CoV-2 antibody responses on serum samples from individuals that had been tested positive after a SARS-CoV-2 outbreak and in control individuals who had a negative PCR result. These analyses were complemented with the determination of spike-antigen specific TH cell responses in the same individuals. All infected participants were presented as asymptomatic or mild cases. Participants initially tested positive for SARS-CoV-2 infection either with PCR, antibody testing, or both had a rapid initial decline in the serum antibody levels in all serological tests but showed a persisting TH cell immunity as assessed by the detection of SARS-CoV-2 specificity of TH cells for up to 1 year after infection. Our data support the notion of a persistent T-cell immunity in mild and asymptomatic cases of SARS-CoV-2 up to 1 year after infection. We show that antibody titers decline over 1 year, but considering several test results, complete seroreversion is rare. Trial registration: German Clinical Trials Register DRKS00022416.

Complicated Long Term Vaccine Induced Thrombotic Immune Thrombocytopenia-A Case Report.

BACKGROUND AND OBJECTIVES: Vaccine induced thrombotic thrombocytopenia (VITT) may occur after COVID-19 vaccination with recombinant adenoviral vector-based vaccines. VITT can present as cerebral sinus and venous thrombosis (CSVT), often complicated by intracranial hemorrhage. Today it is unclear, how long symptomatic VITT can persist. Here, we report the complicated long-term course of a VITT patient with extremely high titers of pathogenic anti-platelet factor 4 (PF4)-IgG antibodies. METHODS: Clinical and laboratory findings are presented, including the course of platelet counts, D-Dimer levels, clinical presentation, imaging, SARS-CoV-2-serological and immunological, platelet activating anti-PF4-IgG, as well as autopsy findings. RESULTS: The patient presented with extended superior sagittal sinus thrombosis with accompanying bifrontal intracerebral hemorrhage. Repeated treatment with intravenous immune globuline (IVIG) resolved recurrent episodes of thrombocytopenia. Moreover, the patient's serum remained strongly positive for platelet-activating anti-PF4-IgG over three months. After a period of clinical stabilization, the patient suffered a recurrent and fatal intracranial hemorrhage. CONCLUSIONS: Complicated VITT with extremely high anti-PF4-IgG titers over three months can induce recurrent thrombocytopenia despite treatment with IVIG and anticoagulation. Plasma exchange, immunoadsorption, and /or immunosuppressive treatment may be considered in complicated VITT to reduce extraordinarily high levels of anti-PF4-IgG. Long-term therapy in such cases must take the individual bleeding risk and CSVT risk into account.

Incidence and severity of G6PI-induced arthritis are not increased in genetically distinct mouse strains upon aging.

BACKGROUND: The incidence of rheumatoid arthritis is correlated with age. In this study, we analyzed the association of the incidence and severity of glucose-6-phosphate isomerase (G6PI)-induced arthritis with age in two different mouse strains. METHODS: Young and very old mice from two different arthritis-susceptible wild-type mouse strains were analyzed after a single subcutaneous injection of G6PI s.c. The metabolism and the function of synoviocytes were analyzed in vitro, the production of bioactive lipid mediators by myeloid cells and synoviocytes was assessed in vitro and ex vivo by UPLC-MS-MS, and flow cytometry was used to verify age-related changes of immune cell composition and function. RESULTS: While the severity of arthritis was independent from age, the onset was delayed in old mice. Old mice showed common signs of immune aging like thymic atrophy associated with decreased CD4+ effector T cell numbers. Despite its decrease, the effector T helper (Th) cell compartment in old mice was reactive and functionally intact, and their Tregs exhibited unaltered suppressive capacities. In homeostasis, macrophages and synoviocytes from old mice produced higher amounts of pro-inflammatory cyclooxygenase (COX)-derived products. However, this functional difference did not remain upon challenge in vitro nor upon arthritis reactions ex vivo. CONCLUSION: While old mice show a higher baseline of inflammatory functions, this does not result in increased reaction towards self-antigens in arthritis-susceptible mouse strains. Together, our data from two different mouse strains show that the susceptibility for G6PI-induced arthritis is not age-dependent.

ATP/IL-33-triggered hyperactivation of mast cells results in an amplified production of pro-inflammatory cytokines and eicosanoids.

IL-33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high-density expression of the IL-33 receptor T1/ST2 (IL-33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL-33 or ATP in damaged peripheral tissues. Whereas IL-33 induces the MyD88-dependent activation of the TAK1-IKK2-NF-κB signalling, ATP induces the Ca2+ -dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro-inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co-sensing) IL-33 and ATP, display an enhanced and prolonged activation of the TAK1-IKK2-NF-κB signalling pathway. This resulted in a massive production of pro-inflammatory cytokines such as IL-2, IL-4, IL-6 and GM-CSF as well as of arachidonic acid-derived cyclooxygenase (COX)-mediated pro-inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co-sensing of ATP and IL-33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE-mediated crosslinking of the FcεRI. Therefore, the IL-33/IL-33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1ß-Induced Mitochondrial Dysfunction in Human Chondrocytes.

Interleukin (IL)-1ß is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1ß-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1ß in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE2, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1ß significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1ß-induced NO release and mitochondrial dysfunction but not IL-1ß-induced release of IL-6, PGE2, and MMP3. Enhancement of cAMP by forskolin reduced IL-1ß-induced NO release and prevented IL-1ß-induced mitochondrial impairment. Activation of AMPK increased IL-1ß-induced NO production and the negative impact of IL-1ß on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1ß-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1ß-induced NO release and mitochondrial dysfunction.

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma.

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.

Candida pathogens induce protective mitochondria-associated type I interferon signalling and a damage-driven response in vaginal epithelial cells.

Vaginal candidiasis is an extremely common disease predominantly caused by four phylogenetically diverse species: Candida albicans; Candida glabrata; Candida parapsilosis; and Candida tropicalis. Using a time course infection model of vaginal epithelial cells and dual RNA sequencing, we show that these species exhibit distinct pathogenicity patterns, which are defined by highly species-specific transcriptional profiles during infection of vaginal epithelial cells. In contrast, host cells exhibit a homogeneous response to all species at the early stages of infection, which is characterized by sublethal mitochondrial signalling inducing a protective type I interferon response. At the later stages, the transcriptional response of the host diverges in a species-dependent manner. This divergence is primarily driven by the extent of epithelial damage elicited by species-specific mechanisms, such as secretion of the toxin candidalysin by C. albicans. Our results uncover a dynamic, biphasic response of vaginal epithelial cells to Candida species, which is characterized by protective mitochondria-associated type I interferon signalling and a species-specific damage-driven response.

Targeting transdifferentiated hepatic stellate cells and monitoring the hepatic fibrogenic process by means of IGF2R-specific peptides designed in silico.

The lack of accurate and easily applicable methods for the diagnosis of liver fibrosis, a disease characterized by an accumulation of the extracellular matrix released by activated hepatic stellate cells (HSCs), has been a major limitation for the clinical management of liver diseases. The identification of biomarkers specific to liver microstructure alterations, combined with a non-invasive optical imaging modality, could guide clinicians towards a therapeutic strategy. In this study, structural information of the insulin-like growth factor 2 receptor (IGF2R), an overexpressed protein on activated HSCs, was used for in silico screening of novel IGF2R-specific peptide ligands. Molecular dynamics simulations, followed by computational alanine scanning of the IGF2R/IGF2 complex, led to the identification of a putative peptide sequence containing the most relevant amino acids for the receptor-ligand interaction (IGF2 E12-C21). The Residue Scan tool, implemented in the MOE software, was then used to optimize the binding affinity of this sequence by amino acid mutations. The designed peptides and their associated scrambled sequences were fluorescently labelled and their binding affinity to LX-2 cells (model for activated human HSCs) was tested using flow cytometry and confocal microscopy. In vitro binding was verified for all sequences (KD ≤ 13.2 µM). With respect to the putative binding sequence, most mutations led to an increased affinity. All sequences have shown superior binding compared to their associated scrambled sequences. Using HPLC, all peptides were tested in vitro for their proteolytic resistance and showed a stability of ≥60% intact after 24 h at 37 °C in 50% v/v FBS. In view of their prospective diagnostic application, a comparison of binding affinity was performed in perpetuated and quiescent-like LX-2 cells. Furthermore, the IGF2R expression for different cell phenotypes was analysed by a quantitative mass spectrometric approach. Our peptides showed increased binding to the perpetuated cell state, indicating their good selectivity for the diagnostically relevant phenotype. In summary, the increased binding affinity of our peptides towards perpetuated LX-2 cells, as well as the satisfactory proteolytic stability, proves that the in silico designed sequences offer a new potential strategy for the targeting of hepatic fibrosis.

IL-3 is essential for ICOS-L stabilization on mast cells, and sustains the IL-33-induced RORγt+ Treg generation via enhanced IL-6 induction.

IL-33 is a member of the IL-1 family. By binding to its receptor ST2 (IL-33R) on mast cells, IL-33 induces the MyD88-dependent activation of the TAK1-IKK2 signalling module resulting in activation of the MAP kinases p38, JNK1/2 and ERK1/2, and of NFκB. Depending on the kinases activated in these pathways, the IL-33-induced signalling is essential for production of IL-6 or IL-2. This was shown to control the dichotomy between RORγt+ and Helios+ Tregs , respectively. SCF, the ligand of c-Kit (CD117), can enhance these effects. Here, we show that IL-3, another growth factor for mast cells, is essential for the expression of ICOS-L on BMMCs, and costimulation with IL-3 potentiated the IL-33-induced IL-6 production similar to SCF. In contrast to the enhanced IL-2 production by SCF-induced modulation of the IL-33 signalling, IL-3 blocked the production of IL-2. Consequently, IL-3 shifted the IL-33-induced Treg dichotomy towards RORγt+ Tregs at the expense of RORγt- Helios+ Tregs . However, ICOS-L expression was downregulated by IL-33. In line with that, ICOS-L did not play any important role in the Treg modulation by IL-3/IL-33-activated mast cells. These findings demonstrate that different from the mast cell growth factor SCF, IL-3 can alter the IL-33-induced and mast cell-dependent regulation of Treg subpopulations by modulating mast cell-derived cytokine profiles.

Antibody response using six different serological assays in a completely PCR-tested community after a coronavirus disease 2019 outbreak-the CoNAN study.

OBJECTIVES: Due to a substantial proportion of asymptomatic and mild courses, many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections remain unreported. Therefore, assessment of seroprevalence may detect the real burden of disease. We aimed to determine and characterize the rate of SARS-CoV-2 infections and the resulting seroprevalence in a defined population. The primary objective of the study was to assess SARS-CoV-2 antibody seroprevalence using six different IgG-detecting immunoassays. Secondary objectives of the study were: (a) to determine potential risk factors for symptomatic versus asymptomatic coronavirus disease 2019 courses, and (b) to investigate the rate of virus RNA-persistence. METHODS: CoNAN is a population-based cohort study performed in the community Neustadt am Rennsteig, Germany, which was quarantined from 22 March to 5 April after six SARS-CoV-2 cases were detected in the village's population. The SARS-CoV-2 outbreak comprised 51 cases and 3 deaths. The CoNAN study was performed from 13 May to 22 May 2020, 6 weeks after a SARS-CoV-2 outbreak. RESULTS: We enrolled a total of 626 participants (71% of the community population) for PCR and antibody testing in the study. All actual SARS-CoV-2 PCR tests were negative. Fifty-two out of 620 (8.4%) participants had antibodies against SARS-CoV-2 in at least two different assays. There were 38 participants with previously PCR-confirmed SARS-CoV-2 infection. Of those, only 19 (50%) displayed anti-SARS-CoV-2 antibodies. We also show that antibody-positive participants with symptoms compatible with a respiratory tract infection had significantly higher antibody levels then asymptomatic participants (EU-assay: median 2.9 versus 7.2 IgG-index, p 0.002; DS-assay: median 45.2 versus 143 AU/mL, p 0.002). Persisting viral replication was not detected. CONCLUSIONS: Our data question the relevance and reliability of IgG antibody testing to detect past SARS-CoV-2 infections 6 weeks after an outbreak. We conclude that assessing immunity for SARS-CoV-2 infection should not rely on antibody tests alone.

The global response to the COVID-19 pandemic: how have immunology societies contributed?

The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The IL-33-induced p38-/JNK1/2-TNFα axis is antagonized by activation of ß-adrenergic-receptors in dendritic cells.

IL-33, an IL-1 cytokine superfamily member, induces the activation of the canonical NF-κB signaling, and of Mitogen Activated Protein Kinases (MAPKs). In dendritic cells (DCs) IL-33 induces the production of IL-6, IL-13 and TNFα. Thereby, the production of IL-6 depends on RelA whereas the production of IL-13 depends on the p38-MK2/3 signaling module. Here, we show that in addition to p65 and the p38-MK2/3 signaling module, JNK1/2 are essential for the IL-33-induced TNFα production. The central roles of JNK1/2 and p38 in DCs are underpinned by the fact that these two MAPK pathways are controlled by activated ß-adrenergic receptors resulting in a selective regulation of the IL-33-induced TNFα response in DCs.