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Monitoring the seroprevalence of SARS-CoV-2 in children and adolescents can provide valuable information for effective SARS-CoV-2 surveillance, and thus guide vaccination strategies. In this study, we quantified antibodies against the spike S1 domains of several SARS-CoV-2 variants (wild-type, Alpha, Delta, and Omicron variants) as well as endemic human coronaviruses (HCoVs) in 1,309 children and adolescents screened between December 2020 and March 2023. Their antibody binding profiles were compared with those of 22 pre-pandemic samples from children and adolescents using an in-house Luminex®-based Corona Array (CA). The primary objectives of this study were to (i) monitor SARS-CoV-2-specific antibodies in children and adolescents, (ii) evaluate whether the S1-specific antibody response can identify the infecting variant of concern (VoC), (iii) estimate the prevalence of silent infections, and (iv) test whether vaccination or infection with SARS-CoV-2 induce HCoV cross-reactive antibodies. Both SARS-CoV-2 infection and vaccination induced a robust antibody response against the S1 domain of WT and VoCs in children and adolescents. Antibodies specific for the S1 domain were able to distinguish between SARS-CoV-2 VoCs in infected children. The serologically identified VoC was typically the predominant VoC at the time of infection. Furthermore, our highly sensitive CA identified more silent SARS-CoV-2 infections than a commercial ELISA (12.1% vs. 6.3%, respectively), and provided insights into the infecting VoC. Seroconversion to endemic HCoVs occurred in early childhood, and vaccination or infection with SARS-CoV-2 did not induce HCoV S1 cross-reactive antibodies. In conclusion, the antibody response to the S1 domain of the spike protein of SARS-CoV-2 is highly specific, providing information about the infecting VoC and revealing clinically silent infections.

BackgroundAtopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis).ObjectiveTo assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp).MethodsTo identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry.ResultsWe identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients’ T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33.ConclusionThe extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

IntroductionCOVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs).MethodsMeasurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay.ResultsThe antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2nd COVID-19 vaccination, but were restored upon the 3rd vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination.DiscussionOverall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.

Abstract Introduction Immunological alterations associated with increased susceptibility to infection are an essential aspect of stroke pathophysiology. Several immunological functions of adipose tissue are altered by obesity and are accompanied by chronic immune activation. The purpose of this study was to examine immune function (monocytes, granulocytes, cytokines) as a function of body mass index (BMI: 1st group: 25; 2nd group: 25 BMI 30; 3rd group: 30) and changes in body weight post stroke. Method Fat status was assessed using standardized weight measurements on days 1, 2, 3, 4, 5, and 7 after ischemic stroke in a cohort of 40 stroke patients and 16 control patients. Liver fat and visceral fat were assessed by MRI on day 1 or 2 [I] and on day 5 or 7 [II]. Leukocyte subpopulations in peripheral blood, cytokines, chemokines, and adipokine concentrations in sera were quantified. In a second cohort (stroke and control group, n = 17), multiple regression analysis was used to identify correlations between BMI and monocyte and granulocyte subpopulations. Results Weight and fat loss occurred from the day of admission to day 1 after stroke without further reduction in the postischemic course. No significant changes in liver or visceral fat were observed between MRI I and MRI II. BMI was inversely associated with IL-6 levels, while proinflammatory cytokines such as eotaxin, IFN-β, IFN -γ and TNF-α were upregulated when BMI increased. The numbers of anti-inflammatory CD14+CD16+ monocytes and CD16+CD62L− granulocytes were reduced in patients with higher BMI values, while that of proinflammatory CD16dimCD62L+ granulocytes was increased. Conclusion A small weight loss in stroke patients was detectable. The data demonstrate a positive correlation between BMI and a proinflammatory poststroke immune response. This provides a potential link to how obesity may affect the clinical outcome of stroke patients.

ABSTRACTThe iron-regulated surface determinant protein B (IsdB) of Staphylococcus aureus is involved in the acquisition of iron from hemoglobin. Moreover, IsdB elicits an adaptive immune response in mice and humans. Here, we show that IsdB also has impact on innate immunity. IsdB induces the release of proinflammatory cytokines, including IL-6 and IL-1β, in innate immune cells of humans and mice. In silico analysis and thermophoresis show that IsdB directly binds to TLR4 with high affinity. TLR4 sensing was essential for the IsdB-mediated production of IL-6, IL-1β, and other cytokines as it was abolished by blocking of TLR4-MyD88-IRAK1/4-NF-κB signaling. The release of IL-1β additionally required activation of the NLRP3 inflammasome. In human monocytes infected with live S. aureus, IsdB was necessary for maximal IL-1β release. Our studies identify S. aureus IsdB as a novel pathogen-associated molecular pattern that triggers innate immune defense mechanisms.IMPORTANCEThe prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1.

The function of T regulatory cells in the tissue fibrosis in chronic pancreatitis is not fully understood. Here the authors use a mouse model of chronic pancreatitis to show that Treg cells reduce IL-4 mediated chronic inflammation in the pancreas associated with M2-like macrophages in vivo.

ObjectiveAcute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life –threatening organ complications, such as respiratory and renal failure, is unknown.DesignOrgan dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification.ResultsSimilar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g-/CD11b+/Ly6c hi monocytes, but not of Ly6g+/CD11b+ neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation.ConclusionIn AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders.

Summary: Background: Understanding how SARS-CoV-2 affects respiratory centres in the brainstem may help to preclude assisted ventilation for patients in intensive care setting. Viral invasion appears unlikely, although autoimmunity has been implicated, the responsible antigens remain unknown. We previously predicted the involvement of three epitopes within distinct brainstem proteins: disabled homolog 1 (DAB1), apoptosis-inducing-factor-1 (AIFM1), and surfeit-locus-protein-1 (SURF1). Methods: Here, we used microarrays to screen serum from COVID-19 patients admitted to intensive care and compared those with controls who experienced mild course of the disease. Findings: The results confirm the occurrence of IgG and IgM antibodies against the hypothesised epitopes in COVID-19 patients. Importantly, while IgM levels were similar in both groups, IgG levels were significantly elevated in severely ill patients compared to controls, suggesting a pathogenic role of IgG. Interpretation: The newly discovered anti-neuronal antibodies might be promising markers of severe disease and the targeted peptide epitopes might be used for targeted immunomodulation. Further work is needed to determine whether these antibodies may play a role in long-COVID. Funding: AF, CF and PR received support from the German Research Foundation (grants FL 379/22-1, 327654276-SFB 1315, FR 4479/1-1, PR 1274/8-1). SH, DR, and DB received support from the Ministry of Economy, State of Mecklenburg Western Pomerania, Germany (grant COVIDPROTECT: “Optimisation of diagnostic and therapeutic pathways for COVID-19 patients in MV”). SH received support from the Research Group Molecular Medicine University of Greifswald (FVMM, seed funding FOVB-2021-01). AV received support from the Else Kröner Fresenius Foundation and the Alzheimer Research Initiative.

Abstract Background Critically ill patients frequently develop muscle atrophy and weakness in the intensive‐care‐unit setting [intensive care unit‐acquired weakness (ICUAW)]. Sepsis, systemic inflammation, and acute‐phase response are major risk factors. We reported earlier that the acute‐phase protein serum amyloid A1 (SAA1) is increased and accumulates in muscle of ICUAW patients, but its relevance was unknown. Our objectives were to identify SAA1 receptors and their downstream signalling pathways in myocytes and skeletal muscle and to investigate the role of SAA1 in inflammation‐induced muscle atrophy. Methods We performed cell‐based in vitro and animal in vivo experiments. The atrophic effect of SAA1 on differentiated C2C12 myotubes was investigated by analysing gene expression, protein content, and the atrophy phenotype. We used the cecal ligation and puncture model to induce polymicrobial sepsis in wild type mice, which were treated with the IкB kinase inhibitor Bristol‐Myers Squibb (BMS)‐345541 or vehicle. Morphological and molecular analyses were used to investigate the phenotype of inflammation‐induced muscle atrophy and the effects of BMS‐345541 treatment. Results The SAA1 receptors Tlr2, Tlr4, Cd36, P2rx7, Vimp, and Scarb1 were all expressed in myocytes and skeletal muscle. Treatment of differentiated C2C12 myotubes with recombinant SAA1 caused myotube atrophy and increased interleukin 6 (Il6) gene expression. These effects were mediated by Toll‐like receptors (TLR) 2 and 4. SAA1 increased the phosphorylation and activity of the transcription factor nuclear factor ‘kappa‐light‐chain‐enhancer' of activated B‐cells (NF‐κB) p65 via TLR2 and TLR4 leading to an increased binding of NF‐κB to NF‐κB response elements in the promoter region of its target genes resulting in an increased expression of NF‐κB target genes. In polymicrobial sepsis, skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with the atrophy response. Inhibition of NF‐κB signalling by BMS‐345541 increased survival (28.6% vs. 91.7%, P < 0.01). BMS‐345541 diminished inflammation‐induced atrophy as shown by a reduced weight loss of the gastrocnemius/plantaris (vehicle: −21.2% and BMS‐345541: −10.4%; P < 0.05), tibialis anterior (vehicle: −22.7% and BMS‐345541: −17.1%; P < 0.05) and soleus (vehicle: −21.1% and BMS‐345541: −11.3%; P < 0.05) in septic mice. Analysis of the fiber type specific myocyte cross‐sectional area showed that BMS‐345541 reduced inflammation‐induced atrophy of slow/type I and fast/type II myofibers compared with vehicle‐treated septic mice. BMS‐345541 reversed the inflammation‐induced atrophy program as indicated by a reduced expression of the atrogenes Trim63/MuRF1, Fbxo32/Atrogin1, and Fbxo30/MuSA1. Conclusions SAA1 activates the TLR2/TLR4//NF‐κB p65 signalling pathway to cause myocyte atrophy. Systemic inhibition of the NF‐κB pathway reduced muscle atrophy and increased survival of septic mice. The SAA1/TLR2/TLR4//NF‐κB p65 atrophy pathway could have utility in combatting ICUAW.

Methicillin-resistant Staphylococcus aureus (MRSA) can colonize dental patients and students, however, studies on the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) among dental health care workers (DHCW) including use of personal protective equipment (PPE) are scarce. We conducted an observational study (StaphDent study) to (I) determine the prevalence of MRSA and MSSA colonization in DHCW in the region of Mecklenburg Western-Pomerania, Germany, (II) resolve the S. aureus population structure to gain hints on possible transmission events between co-workers, and (III) clarify use of PPE. Nasal swabs were obtained from dentists (n = 149), dental assistants (n = 297) and other dental practice staff (n = 38). Clonal relatedness of MSSA isolates was investigated using spa typing and, in some cases, whole genome sequencing (WGS). PPE use was assessed by questionnaire. While 22.3% (108/485) of the participants were colonized with MSSA, MRSA was not detected. MSSA prevalence was not associated with size of dental practices, gender, age, or duration of employment. The identified 61 spa types grouped into 17 clonal complexes and four sequence types. Most spa types (n = 47) were identified only once. In ten dental practices one spa type occurred twice. WGS data analysis confirmed a close clonal relationship for 4/10 isolate pairs. PPE was regularly used by most dentists and assistants. To conclude, the failure to recover MRSA from DHCW reflects the low MRSA prevalence in this region. Widespread PPE use suggests adherence to routine hygiene protocols. Compared to other regional HCW MRSA rates the consequent usage of PPE seems to be protective.

Abstract Background Dialysis patients are frequently exposed to Staphylococcus aureus due to stays in dialysis centers, hospitals or rest homes. The hemodialysis vascular access is a potential entry site for S. aureus, in particular when using a central venous catheter (CVC) which increases the risk of sepsis compared to arteriovenous (AV) fistula. We prospectively followed a cohort of 86 hemodialysis patients from an outpatient dialysis center over 25 months analyzing S. aureus carrier status, S. aureus infection rates and mortality. Methods Demographic data and patients´ medical histories were collected and followed from all hemodialysis patients. Blood samples, nasal swabs and swabs from the hemodialysis vascular access site were taken every six months for a period of 25 months and tested for S. aureus. Strains were cultured and further characterized by spa PCR and microarray-based genotyping. Resulting data were compared with those from the general population. Results In cross-sectional analyses, an average of 40% of hemodialysis patients were S. aureus carriers compared to 27% in the general population. Longitudinally, a total of 65% were S. aureus carriers: 16% were persistent carriers, 43% were intermittently colonized. The most common S. aureus lineage in the dialysis patient cohort was the clonal complex (CC) 8 and the spa type t008, while in the general population, the clonal complex CC30 dominates. During the study period, we observed six S. aureus-associated blood stream infections with one S. aureus attributable death. S. aureus carriers with an AV fistula were more densely colonized in the nasal mucosa compared to patients with a CVC. Overall mortality was lower for hemodialysis patients with a positive S. aureus carrier status compared to non-carriers (hazard ratio of 0.19). Conclusions Compared to the general population, hemodialysis patients were more frequently colonized with S. aureus and displayed both different S. aureus colonization densities as well as lineages, possibly explained by more frequent exposure to health care environments. The lower overall mortality in carriers compared to non-carriers is intriguing and will be investigated in detail in the future. Trial registration ISRCTN 14385893, 2. October 2018, retrospectively registered.

Abstract Reactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown. Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs oxOva). Dynamic light scattering and circular dichroism spectroscopy reveal structural modifications in oxOva compared to Ova. T cells from Ova‐specific OT‐II but not from C57BL/6 or SKH‐1 wild type mice presents enhanced activation after Ova addition. OxOva exacerbates this activation when administered ex vivo or in vivo, along with an increased interferon‐gamma production, a known anti‐melanoma agent. OxOva vaccination of wild type mice followed by inoculation of syngeneic B16F10 Ova‐expressing melanoma cells shows enhanced T cell number and activation, decreased tumor burden, and elevated numbers of antigen‐presenting cells when compared to their Ova‐vaccinated counterparts. Analysis of oxOva using mass spectrometry identifies three hot spots regions rich in oxidative modifications that are associated with the increased T cell activation. Using Ova as a model protein, the findings suggest an immunomodulating role of multi‐ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.

Protection against Staphylococcus aureus is determined by the polarization of the anti-bacterial immune effector mechanisms. Virulence factors of S. aureus can modulate these and induce differently polarized immune responses in a single individual. We proposed that this may be due to intrinsic properties of the bacterial proteins. To test this idea, we selected two virulence factors, the serine protease-like protein B (SplB) and the glycerophosphoryl diester phosphodiesterase (GlpQ). In humans naturally exposed to S. aureus, SplB induces a type 2-biased adaptive immune response, whereas GlpQ elicits type 1/type 3 immunity. We injected the recombinant bacterial antigens into the peritoneum of S. aureus-naïve C57BL/6N mice and analyzed the immune response. This was skewed by SplB toward a Th2 profile including specific IgE, whereas GlpQ was weakly immunogenic. To elucidate the influence of adjuvants on the proteins’ polarization potential, we studied Montanide ISA 71 VG and Imject™Alum, which promote a Th1 and Th2 response, respectively. Alum strongly increased antibody production to the Th2-polarizing protein SplB, but did not affect the response to GlpQ. Montanide enhanced the antibody production to both S. aureus virulence factors. Montanide also augmented the inflammation in general, whereas Alum had little effect on the cellular immune response. The adjuvants did not override the polarization potential of the S. aureus proteins on the adaptive immune response.

Our goal was to provide a comprehensive overview of the antibody response to Staphylococcus aureus antigens in the general population as a basis for defining disease-specific profiles and diagnostic signatures. We tested the specific IgG and IgA responses to 79 staphylococcal antigens in 996 individuals from the population-based Study of Health in Pomerania. Using a dilution-based multiplex suspension array, we extended the dynamic range of specific antibody detection to seven orders of magnitude, allowing the precise quantification of high and low abundant antibody specificities in the same sample. The observed IgG and IgA antibody responses were highly heterogeneous with differences between individuals as well as between bacterial antigens that spanned several orders of magnitude. Some antigens elicited significantly more IgG than IgA and vice versa. We confirmed a strong influence of colonization on the antibody response and quantified the influence of sex, smoking, age, body mass index, and serum glucose on anti-staphylococcal IgG and IgA. However, all host parameters tested explain only a small part of the extensive variability in individual response to the different antigens of S. aureus.

In cystic fibrosis (CF) infectious and allergic airway inflammation cause pulmonary exacerbations that destroy the lungs. Staphylococcus aureus is a common long-term colonizer and cause of recurrent airway infections in CF. The pathogen is also associated with respiratory allergy; especially the staphylococcal serine protease-like proteins (Spls) can induce type 2 immune responses in humans and mice. We measured the serum IgE levels specific to 7 proteases of S. aureus by ELISA, targeting 5 Spls (76 CF patients and 46 controls) and the staphopains A and B (16 CF patients and 46 controls). Then we compared cytokine release and phenotype of T cells that had been stimulated with Spls between 5 CF patients and 5 controls. CF patients had strongly increased serum IgE binding to all Spls but not to the staphopains. Compared to healthy controls, their Spl-stimulated T cells released more type 2 cytokines (IL-4, IL-5, IL-13) and more IL-6 with no difference in the secretion of type 1- or type 3 cytokines (IFNγ, IL-17A, IL-17F). IL-10 production was low in CF T cells. The phenotype of the Spl-exposed T cells shifted towards a Th2 or Th17 profile in CF but to a Th1 profile in controls. Sensitization to S. aureus Spls is common in CF. This discovery could explain episodes of allergic inflammation of hitherto unknown causation in CF and extend the diagnostic and therapeutic portfolio.

The monoclonal antibody against CTLA-4, Ipilimumab, is a first-in-class immune-checkpoint inhibitor approved for treatment of advanced melanoma in adults but not extensively studied in children. In light of the fact that the immune response early in life differs from that of adults, we have applied a human in vitro model stimulating CD4+ T-cells from neonates, children (1–5 years), and adults antigen-specifically with Staphylococcus aureus (S. aureus) for assessment of CTLA-4 blockade early in life. We show that T-cell proliferation as well as frequencies of antigen-specific T-cells (CD40L+CD4+) were enhanced in neonatal T-cells upon CTLA-4 blockade showing a larger variance within the group (F-test p

Background: Stroke patients are at risk of acquiring secondary infections due to stroke-induced immune suppression (SIIS). Immunosuppressive cells comprise myeloid-derived suppressor cells (MDSCs) and immunosuppressive interleukin 10 (IL-10)-producing monocytes. MDSCs represent a small but heterogeneous population of monocytic, polymorphonuclear (or granulocytic), and early progenitor cells (“early” MDSC), which can expand extensively in pathophysiological conditions. MDSCs have been shown to exert strong immune-suppressive effects. The role of IL-10-producing immunosuppressive monocytes after stroke has not been investigated, but monocytes are impaired in oxidative burst and downregulate human leukocyte antigen—DR isotype (HLA-DR) on the cell surface.Objectives: The objective of this work was to investigate the regulation and function of MDSCs as well as the immunosuppressive IL-10-producing monocytes in experimental and human stroke.Methods: This longitudinal, monocentric, non-interventional prospective explorative study used multicolor flow cytometry to identify MDSC subpopulations and IL-10 expression in monocytes in the peripheral blood of 19 healthy controls and 27 patients on days 1, 3, and 5 post-stroke. Quantification of intracellular STAT3p and Arginase-1 by geometric mean fluorescence intensity was used to assess the functionality of MDSCs. In experimental stroke induced by electrocoagulation in middle-aged mice, monocytic (CD11b+Ly6G−Ly6Chigh) and polymorphonuclear (CD11b+Ly6G+Ly6Clow) MDSCs in the spleen were analyzed by flow cytometry.Results: Compared to the controls, stroke patients showed a relative increase in monocytic MDSCs (percentage of CD11b+ cells) in whole blood without evidence for an altered function. The other MDSC subgroups did not differ from the control. Also, in experimental stroke, monocytic, and in addition, polymorphonuclear MDSCs were increased. The numbers of IL-10-positive monocytes did not differ between the patients and controls. However, we provide a new insight into monocytic function post-stroke since we can report that a differential regulation of HLA-DR and PD-L1 was found depending on the IL-10 production of monocytes. IL-10-positive monocytes are more activated post-stroke, as indicated by their increased HLA-DR expression.Conclusions: MDSC and IL-10+ monocytes can induce immunosuppression within days after stroke.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.

In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis.

Although antigen-specific priming of antibody responses is impaired during sepsis, there is nevertheless a strong increase in IgM and IgG serum concentrations. Using colon ascendens stent peritonitis (CASP), a mouse model of polymicrobial abdominal sepsis, we observed substantial increases in IgM as well as IgG of all subclasses, starting at day 3 and peaking 2 weeks after sepsis induction. The dominant source of antibody-secreting cells was by far the spleen, with a minor contribution of the mesenteric lymph nodes. Remarkably, sepsis induction in splenectomized mice did not change the dynamics of the serum IgM/IgG reaction, indicating that the marginal zone B cells, which almost exclusively reside in the spleen, are dispensable in such a setting. Hence, in systemic bacterial infection, the function of the spleen as dominant niche of antibody-producing cells can be compensated by extra-splenic B cell populations as well as other lymphoid organs. Depletion of CD4+ T cells did not affect the IgM response, while it impaired IgG generation of all subclasses with the exception of IgG3. Taken together, our data demonstrate that the robust class-switched antibody response in sepsis encompasses both T cell-dependent and -independent components.

ABSTRACT Systemic and quantitative investigations of human plasma proteins (proteomics) and Staphylococcus aureus-specific antibodies (immunoproteomics) provide complementary information and hold promise for the discovery of biomarkers in Staphylococcus aureus bloodstream infection (SABSI). Usually, data-dependent acquisition (DDA) is used for proteome analysis of serum or plasma, but data-independent acquisition (DIA) is more comprehensive and reproducible. In this prospective cohort study, we aimed to identify biomarkers associated with the early stages of SABSI using a serum DIA proteomic and immunoproteomic approach. Sera from 49 SABSI patients and 43 noninfected controls were analyzed. In total, 608 human serum proteins were identified with DIA. A total of 386 proteins could be quantified, of which 9 proteins, mainly belonging to acute-phase proteins, were significantly increased, while 7 high-density lipoproteins were lower in SABSI. In SABSI, total anti-S. aureus serum IgG was reduced compared with controls as shown by immunoproteomic quantification of IgG binding to 143 S. aureus antigens. IgG binding to 48 of these anti-S. aureus proteins was significantly lower in SABSI, while anti-Ecb IgG was the only one increased in SABSI. Serum IgG binding to autoinducing peptide MsrB, FadB, EsxA, Pbp2, FadB, SspB, or SodA was very low in SABSI. This marker panel discriminated early SABSI from controls with 95% sensitivity and 100% specificity according to random forest prediction. This holds promise for patient stratification according to their risk of S. aureus infection, underlines the protective function of the adaptive immune system, and encourages further efforts in the development of a vaccine against S. aureus. IMPORTANCE S. aureus sepsis has a high complication and mortality rate. Given the limited therapeutic possibilities, effective prevention strategies, e.g., a vaccine, or the early identification of high-risk patients would be important but are not available. Our study showed an acute-phase response in patients with S. aureus bloodstream infection and evidence that lipoproteins are downregulated in plasma. Using immunoproteomics, stratification of patients appears to be achievable, since at the early stages of systemic S. aureus infection patients had low preexisting anti-S. aureus antibody levels. This strengthens the notion that a robust immune memory for S. aureus protects against infections with the pathogen.

Abstract Background Stroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE. Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here. Methods Ex vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy. Results MPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release. r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA. Conclusions Intracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies. r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.

Due to increasing mupirocin resistance, alternatives for Staphylococcus aureus nasal decolonization are urgently needed. Adhesion inhibitors are promising new preventive agents that may be less prone to induce resistance, as they do not interfere with the viability of S. aureus and therefore exert less selection pressure. We identified promising adhesion inhibitors by screening a library of 4208 compounds for their capacity to inhibit S. aureus adhesion to A-549 epithelial cells in vitro in a novel automated, imaging-based assay. The assay quantified DAPI-stained nuclei of the host cell; attached bacteria were stained with an anti-teichoic acid antibody. The most promising candidate, aurintricarboxylic acid (ATA), was evaluated in a novel persistent S. aureus nasal colonization model using a mouse-adapted S. aureus strain. Colonized mice were treated intranasally over 7 days with ATA using a wide dose range (0.5–10%). Mupirocin completely eliminated the bacteria from the nose within three days of treatment. In contrast, even high concentrations of ATA failed to eradicate the bacteria. To conclude, our imaging-based assay and the persistent colonization model provide excellent tools to identify and validate new drug candidates against S. aureus nasal colonization. However, our first tested candidate ATA failed to induce S. aureus decolonization.

We asked whether transient Staphylococcus aureus in the oral environment synergistically interacts with orally associated bacterial species such as Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar (six-species control biofilm 6S). For this purpose, four modified biofilms with seven species that contain either the wild type strain of the S. aureus genotype (USA300-MRSA WT), its isogenic mutant with MSCRAMM deficiency (USA300-MRSA ΔMSCRAMM), a methicillin-sensitive S. aureus (ST72-MSSA-) or a methicillin-resistant S. aureus (USA800-MRSA) grown on hydroxyapatite disks were examined. Culture analyses, confocal-laser-scanning microscopy and proteome analyses were performed. S. aureus strains affected the amount of supragingival biofilm-associated species differently. The deletion of MSCRAMM genes disrupted the growth of S. aureus and the distribution of S. mutans and S. oralis within the biofilms. In addition, S. aureus caused shifts in the number of detectable proteins of other species in the 6S biofilm. S. aureus (USA300-MRSA WT), aggregated together with early colonizers such as Actinomyces and streptococci, influenced the number of secondary colonizers such as Fusobacterium nucleatum and was involved in structuring the biofilm architecture that triggered the change from a homeostatic biofilm to a dysbiotic biofilm to the development of oral diseases.

To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP) encoding a continuously expressed green fluorescent protein (GFP). Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed). Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC) standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]). They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset. Keywords: Epithelial cells, Flow cytometry, Internalization, Host-pathogen interaction, Proteomics, SILAC, Staphylococcus aureus

Staphylococcus aureus superantigens (SAgs) are among the most potent T cell mitogens known. They stimulate large fractions of T cells by cross-linking their T cell receptor with major histocompatibility complex class-II molecules on antigen presenting cells, resulting in T cell proliferation and massive cytokine release. To date, 26 different SAgs have been described in the species S. aureus; they comprise the toxic shock syndrome toxin (TSST-1), as well as 25 staphylococcal enterotoxins (SEs) or enterotoxin-like proteins (SEls). SAgs can cause staphylococcal food poisoning and toxic shock syndrome and contribute to the clinical symptoms of staphylococcal infection. In addition, there is growing evidence that SAgs are involved in allergic diseases. This review provides an overview on recent epidemiological data on the involvement of S. aureus SAgs and anti-SAg-IgE in allergy, demonstrating that being sensitized to SEs—in contrast to inhalant allergens—is associated with a severe disease course in patients with chronic airway inflammation. The mechanisms by which SAgs trigger or amplify allergic immune responses, however, are not yet fully understood. Here, we discuss known and hypothetical pathways by which SAgs can drive an atopic disease.

Whether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. We previously identified a mouse-adapted S. aureus strain, which caused infections in laboratory mice. This raised the question whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments. Publicly available health reports from commercial vendors revealed that S. aureus colonization is rather frequent, with rates as high as 21% among specific-pathogen-free mice. In animal facilities, S. aureus was readily transmitted from parents to offspring, which became persistently colonized. Among 99 murine S. aureus isolates from Charles River Laboratories half belonged to the lineage CC88 (54.5%), followed by CC15, CC5, CC188, and CC8. A comparison of human and murine S. aureus isolates revealed features of host adaptation. In detail, murine strains lacked hlb-converting phages and superantigen-encoding mobile genetic elements, and were frequently ampicillin-sensitive. Moreover, murine CC88 isolates coagulated mouse plasma faster than human CC88 isolates. Importantly, S. aureus colonization clearly primed the murine immune system, inducing a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates. Phospholipase C emerged as a promising test antigen for monitoring S. aureus colonization in laboratory mice. In conclusion, laboratory mice are natural hosts of S. aureus and therefore, could provide better infection models than previously assumed. Pre-exposure to the bacteria is a possible confounder in S. aureus infection and vaccination studies and should be monitored.

Background and Purpose. Regulatory T cells (Tregs) have been suggested to modulate stroke-induced immune responses. However, analyses of Tregs in patients and in experimental stroke have yielded contradictory findings. We performed the current study to assess the regulation and function of Tregs in peripheral blood of stroke patients. Age dependent expression of CD39 on Tregs was quantified in mice and men. Methods. Total FoxP3+ Tregs and CD39+FoxP3+ Tregs were quantified by flow cytometry in controls and stroke patients on admission and on days 1, 3, 5, and 7 thereafter. Treg function was assessed by quantifying the inhibition of activation-induced expression of CD69 and CD154 on T effector cells (Teffs). Results. Total Tregs accounted for 5.0% of CD4+ T cells in controls and

Staphylococcus aureus (S. aureus) is a dangerous pathogen as well as a frequent colonizer, threatening human health worldwide. Protection against S. aureus infection is challenging, as the bacteria have sophisticated strategies to escape the host immune response. To maintain equilibrium with S. aureus, both innate and adaptive immune effector mechanisms are required. Dendritic cells (DCs) are critical players at the interface between the two arms of the immune system, indispensable for inducing specific T cell responses. In this review, we highlight the importance of DCs in mounting innate as well as adaptive immune responses against S. aureus with emphasis on their role in S. aureus-induced respiratory diseases. We also review what is known about mechanisms that S. aureus has adopted to evade DCs or manipulate these cells to its advantage.

Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms of protection will be key for the successful prevention or treatment of S. aureus invasion. Omics technologies generate a comprehensive picture of the physiological and pathophysiological processes within cells, tissues, organs, organisms and even populations. This review provides an overview of the contribution of genomics, transcriptomics, proteomics, metabolomics and immunoproteomics to the current understanding of S. aureus‑host interaction, with a focus on the adaptive immune response to the microorganism. While antibody responses during colonization and infection have been analyzed in detail using immunoproteomics, the full potential of omics technologies has not been tapped yet in terms of T-cells. Omics technologies promise to speed up vaccine development by enabling reverse vaccinology approaches. In consequence, omics technologies are powerful tools for deepening our understanding of the “superbug” S. aureus and for improving its control.

Staphylococcus aureus (S. aureus) is a dangerous pathogen and a leading cause of both nosocomial and community acquired bacterial infection worldwide. However, on the other hand, we are all exposed to this bacterium, often within the first hours of life, and usually manage to establish equilibrium and coexist with it. What does the adaptive immune system contribute toward lifelong control of S. aureus? Will it become possible to raise or enhance protective immune memory by vaccination? While in the past the S. aureus-specific antibody response has dominated this discussion, the research community is now coming to appreciate the role that the cellular arm of adaptive immunity, the T cells, plays. There are numerous T cell subsets, each with differing functions, which together have the ability to orchestrate the immune response to S. aureus and hence to tip the balance between protection and pathology. This review summarizes the state of the art in this dynamic field of research.