•Functional activity of C3 and levels of initiation factors are similar in male and female mice.•Functional activity of C9, and serum levels of C6 and C9 is very low in female mice.•Gender dimorphism ...is present in C57BL/6 and BALB/c mice, but not in CD-1 Swiss mice.
Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9.
ABSTRACT
The complement system, and specifically C5a, is involved in renal ischemia‐reperfusion (IR) injury. The 2 receptors for complement anaphylatoxin C5a (C5aR1 and C5aR2) are expressed on ...leukocytes as well as on renal epithelium. Extensive evidence shows that C5aR1 inhibition protects kidneys from IR injury; however, the role of C5aR2 in IR injury is less clear as initial studies proposed the hypothesis that C5aR2 functions as a decoy receptor. By Using wild‐type, C5aR1‐/‐, and C5aR2‐/‐ mice in a model of renal IR injury, we found that a deficiency of either of these receptors protected mice from renal IR injury. Surprisingly, C5aR2‐/‐ mice were most protected and had lower creatinine levels and reduced acute tubular necrosis. Next, an in vivo migration study demonstrated that leukocyte chemotaxis was unaffected in C5aR2‐/‐ mice, whereas neutrophil activation was reduced by C5aR2 deficiency. To further investigate the contribution of renal cell‐expressed C5aR2 vs. leukocyte‐expressed C5aR2 to renal IR injury, bone marrow chimeras were created. Our data show that both renal cell‐expressed C5aR2 and leukocyte‐expressed C5aR2 mediate IR‐induced renal dysfunction. These studies reveal the importance of C5aR2 in renal IR injury. They further show that C5aR2 is a functional receptor, rather than a decoy receptor, and may provide a new target for intervention.—Poppelaars, F., van Werkhoven, M. B., Kotimaa, J., Veldhuis, Z. J., Ausema, A., Broeren, S. G. M., Damman, J., Hempel, J. C., Leuvenink, H. G. D., Daha, M. R., van Son, W. J., van Kooten, C., van Os, R. P., Hillebrands, J.‐L., Seelen, M. A. Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia‐reperfusion injury. FASEB J. 31, 3193–3204 (2017). www.fasebj.org
The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also ...contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24h following reperfusion, which returned to base-line at 3 and 7days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.
Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N‐maleimide‐cored ...polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4‐conjugate addition to give one‐to‐one protein–polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single‐chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein–dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (λ≈350 nm) can cleave the photolabile o‐nitrobenzyl‐linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.
Glue for proteins and DNA: Experimental studies and molecular dynamics modeling demonstrates that multivalent dendrons can be utilized to temporarily glue proteins and DNA together with high affinity (see figure). UV exposure can degrade the photolabile surface of the dendron, which results in loss of the multivalent binding interactions and release of the DNA and protein.
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Preeclampsia is a serious vascular complication of the human pregnancy, whose etiology is still poorly understood. In preeclampsia, exacerbated apoptosis and fragmentation of the ...placental tissue occurs due to developmental qualities of the placental trophoblast cells and/or mechanical and oxidative distress to the syncytiotrophoblast, which lines the placental villi. Dysregulation of the complement system is recognized as one of the mechanisms of the disease pathology. Complement has the ability to promote inflammation and facilitate phagocytosis of placenta-derived particles and apoptotic cells by macrophages. In preeclampsia, an overload of placental cell damage or dysregulated complement system may lead to insufficient clearance of apoptotic particles and placenta-derived debris. Excess placental damage may lead to sequestration of microparticles, such as placental vesicles, to capillaries in the glomeruli of the kidney and other vulnerable tissues. This phenomenon could contribute to the manifestations of typical diagnostic symptoms of preeclampsia: proteinuria and new-onset hypertension. In this review we propose that the complement system may serve as a regulator of the complex tolerance and clearance processes that are fundamental in healthy pregnancy. It is therefore recommended that further research be conducted to elucidate the interactions between components of the complement system and immune responses in the context of complicated and healthy pregnancy.
Background and Aim
Single‐cell RNA sequencing (scRNA‐seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of ...oligonucleotide‐labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA‐seq‐only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co‐functioning oligonucleotide‐labelled antibodies.
Main Methods and Results
We present here a simple adjustable oligonucleotide‐antibody conjugation method which enables a desired level of oligo‐conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA‐seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo‐antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA‐seq.
Conclusions and Implications
Optimised oligo‐labelling will improve detection of weak protein targets in scRNA‐seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE‐seq libraries. Furthermore, the use of a pre‐stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA‐seq multimodal experiments.
Graphical and Lay Summary
Antibodies labelled with oligo‐DNA strands are currently utilised in, for example, multimodal single‐cell RNA sequencing and highly multiplexed in situ staining protocols. In this study the authors have developed and tested a method where it is easy to modify the number of oligos attached to a single antibody molecule, which enhances the detection of low expression protein targets. This conjugation method is also compatible with different cell fixations methods.
Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is the most common tickborne disease. Its neuronal form, neuroborreliosis, comprises 3 to 38% of borreliosis cases in Europe. Borrelia ...outer surface proteins and virulence factors, OspE and BBK32, have been previously reported to help cause infection by promoting attachment to human host epithelial cells and evading complement attack. We assessed the serological responses to BBK32 and OspE in 19 individuals diagnosed with neuroborreliosis to see whether antibodies that could both target the bacteria and neutralize the virulence mechanisms on the microbial surface emerge. Results evaluate levels of total protein, IgG and the chemokine CXCL13, a determinant for B‐cell recruitment during neuroinflammation, in patients' cerebrospinal fluid samples. Antibody levels against BBK32 and OspE correlated with those against VlsE, a well‐characterized diagnostic serological marker of the disease. A dual serological profile of the patients was observed. K‐means clustering split the cohort into two discrete groups presenting distinct serological and CNS responses. One group contained young patients with low levels of anti‐BBK32 and OspE antibodies. The other group showed stronger responses, possibly following prolonged infections or reinfections. Additionally, we assessed anti‐ganglioside antibodies that could cause autoimmunity or complement dysregulation but observed that they did not correlate with neuroborreliosis in our patient cohort. The dual nature of antibody responses against the virulence factors BBK32 and OspE in neuroborreliosis patients may suggest the necessity of repeated exposures for efficient immune responses. Better protection could be achieved if the virulence factors were formulated into vaccines.
In our study, we observed that Borrelia bacteria express virulence factors OspE and BBK32, indicated by the serological responses detected in patients with neuroborreliosis. The patient cohort clustered into two groups based on their serological responses and inflammatory marker levels. The serological responses were not sufficient to prevent the infection.
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