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Objective
Acute 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.
Design
Organ 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.
Results
Similar 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.
Conclusion
In 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.
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.
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.
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.
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.
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.
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.