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Abstract
Mechanical characteristics of individual cells play a vital role in many biological processes and are considered as indicators of the cellsâ states. Disturbances including methylâÎČâcyclodextrin (MÎČCD) and cytochalasin D (cytoD) are known to significantly affect the state of cells, but little is known about the realâtime response of single cells to these drugs in their physiological condition. Here, nanoindentationâbased atomic force microscopy (AFM) was used to measure the elasticity of human embryonic kidney cells in the presence and absence of these pharmaceuticals. The results showed that depletion of cholesterol in the plasma membrane with MÎČCD resulted in cell stiffening whereas depolymerization of the actin cytoskeleton by cytoD resulted in cell softening. Using AFM for realâtime measurements, we observed that cells mechanically responded right after these drugs were added. In more detail, the cellÂŽs elasticity suddenly increased with increasing instability upon cholesterol extraction while it is rapidly decreased without changing cellular stability upon depolymerizing actin cytoskeleton. These results demonstrated that actin cytoskeleton and cholesterol contributed differently to the cell mechanical characteristics.
Abstract
Background
Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neoâepitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcÎłRIIA, release bactericidal substances, and kill Gramânegative Escherichia coli.
Objectives
To assess the role of PF4, antiâPF4/H antibodies and FcÎłRIIa in killing of Gramâpositive bacteria by platelets.
Methods
Binding of PF4 to proteinâA deficient Staphylococcus aureus (SA113Îspa) and nonâencapsulated Streptococcus pneumoniae (D39Îcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived antiâPF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62Pâexpression, PACâ1 binding) and phosphatidylserine (PS)âexposure were analyzed.
Results
Monoclonal and patientâderived antiâPF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6âfold increased fluorescence signal for human antiâPF4/H antibodies to 24.0âfold increase for KKO). Staphylococcus aureus (5.5 Ă 104cfu/mL) was efficiently killed by platelets (2.7 Ă 104cfu/mL) or their releasate (2.9 Ă 104cfu/mL). Killing was not further enhanced by PF4 or antiâPF4/H antibodies. Blocking FcÎłRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAPâ6 stimulation and exposed high levels of PS.
Conclusions
AntiâPF4/H antibodies seem to have only a minor role for direct killing of Gramâpositive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.
Abstract
Background
Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparinâinduced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6â, 8â, 10â, and 12âmer size and a hypersulfated 12âmer (S12âmer).
Methods
We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and antiâPF4/heparin antibodies.
Results
The synthetic heparinâlike compounds display stronger binding characteristics to PF4 than animalâderived heparins of corresponding lengths. Upon complexation with PF4, 6âmer and S12âmer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8â, 10â, and 12âmer heparins. AntiâPF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins â€Â 8âmer than with complexes formed between PF4 and heparins â„ 10âmer. Addition of one sulfate group to the 12âmer resulted in a S12âmer, which showed substantial changes in its binding characteristics to PF4.
Conclusions
We provide a template for characterizing interactions of newly developed heparinâbased anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.
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 γΎ 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.
Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO2) are the key enzymes of tryptophan (TRP) metabolism in the kynurenine pathway (KP). Both enzymes function as indicators of immunosuppression and poor survival in cancer patients. Direct or indirect targeting of either of these substances seems thus reasonable to improve therapy options for patients. In this study, glioblastoma multiforme (GBM) as well as head and neck squamous cell carcinomas (HNSCC) were examined because of their different mechanisms of spontaneous and treatment-induced immune escape. Effects on gene expression and protein levels were examined. Accompanying assessment of TRP metabolites from treated GBM cell culture supernatants was conducted. Our results show a heterogeneous and inversely correlated expression profile of TRP-metabolizing genes among GBM and HNSCC cells, with low, but inducible IDO1 expression upon IFNÎł treatment. TDO2 expression was higher in GBM cells, while genes encoding kynurenine aminotransferases were mainly confined to HNSCC cells. These data indicate that the KP is active in both entities, with however different enzymes involved in TRP catabolism. Upon treatment with Temozolomide, the standard of care for GBM patients, IDO1 was upregulated. Comparable, although less pronounced effects were seen in HNSCC upon Cetuximab and conventional drugs (i.e., 5-fluorouracil, Gemcitabine). Here, IDO1 and additional genes of the KP (KYAT1, KYAT2, and KMO) were induced. Vice versa, the novel yet experimental cyclin-dependent kinase inhibitor Dinaciclib suppressed KP in both entities. Our comprehensive data imply inhibition of the TRP catabolism by Dinaciclib, while conventional chemotherapeutics tend to activate this pathway. These data point to limitations of conventional therapy and highlight the potential of targeted therapies to interfere with the cells' metabolism more than anticipated.
Background: Postoperatively acquired immune dysfunction is associated with a higher mortality rate in case of septic complications. As details of this severe clinical problem are still unknown, animal models are essential to characterise the mechanisms involved. Methods: Mice were laparotomised and the small intestine was pressed smoothly in antegrade direction. For extension of trauma, the intestine was manipulated three times consecutively. Following this, the ex vivo cytokine release of splenocytes was determined. The degree of surgical trauma was analysed by detection of HMGB1 and IL-6 in serum and by neutrophil staining in the muscularis mucosae. Results: We adapted the previously described animal model of intestinal manipulation to provide a model of surgically induced immune dysfunction. Following intestinal manipulation, the mice showed elevated serum levels of HMGB1 and IL-6 and increased infiltration of granulocytes into the muscularis mucosae. Ex vivo cytokine release by splenocytes was suppressed in the postoperative period. The degree of suppression correlated with the extent of surgical trauma. Conclusions: In this study, we describe a surgically induced immune dysfunction animal model, in which a significant surgical trauma is followed by an immune dysfunction. This model may be ideal for the characterisation of the postoperative immune dysfunction syndrome.
