Evidence, detailed characterization and clinical context of complement activation in acute multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C) is a rare, life-threatening complication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. MIS-C develops with high fever, marked inflammation and shock-like picture several weeks after exposure to, or mild infection with SARS-CoV-2. Deep immune profiling identified activated macrophages, neutrophils, B-plasmablasts and CD8 + T cells as key determinants of pathogenesis together with multiple inflammatory markers. The disease rapidly responds to intravenous immunoglobulin (IVIG) treatment with clear changes of immune features. Here we present the results of a comprehensive analysis of the complement system in the context of MIS-C activity and describe characteristic changes during IVIG treatment. We show that activation markers of the classical, alternative and terminal pathways are highly elevated, that the activation is largely independent of anti-SARS-CoV-2 humoral immune response, but is strongly associated with markers of macrophage activation. Decrease of complement activation is closely associated with rapid improvement of MIS-C after IVIG treatment

MASP-1 Increases Endothelial Permeability

Pathologically increased vascular permeability is an important dysfunction in the pathomechanism of life-threatening conditions, such as sepsis, ischemia/reperfusion, or hereditary angioedema (HAE), diseases accompanied by uncontrolled activation of the complement system. HAE for example is caused by the deficiency of C1-inhibitor (the main regulator of early complement activation), which leads to edematous attacks threatening with circulatory collapse. We have previously reported that endothelial cells become activated during HAE attacks. A natural target of C1-inhibitor is mannan-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, which plays a key role in the activation of complement lectin pathway. We have previously shown that MASP-1 induces the pro-inflammatory activation of endothelial cells and in this study we investigated whether MASP-1 can directly affect endothelial permeability. All experiments were performed on human umbilical vein endothelial cells (HUVECs). Real-time micro electric sensing revealed that MASP-1 decreases the impedance of HUVEC monolayers and in a recently developed permeability test (XperT), MASP-1 dose-dependently increased endothelial paracellular transport. We show that protease activated receptor-1 mediated intracellular Ca2+-mobilization, Rho-kinase activation dependent myosin light chain (MLC) phosphorylation, cytoskeletal actin rearrangement, and disruption of interendothelial junctions are underlying this phenomenon. Furthermore, in a whole-transcriptome microarray analysis MASP-1 significantly changed the expression of 25 permeability-related genes in HUVECs—for example it up-regulated bradykinin B2 receptor expression. According to our results, MASP-1 has potent permeability increasing effects. During infections or injuries MASP-1 may help eliminate the microbes and/or tissue debris by enhancing the extravasation of soluble and cellular components of the immune system, however, it may also play a role in the pathomechanism of diseases, where edema formation and complement lectin pathway activation are simultaneously present. Our findings also raise the possibility that MASP-1 may be a promising target of anti-edema drug development

Serum MASP-1 in complex with MBL activates endothelial cells.

The complement system plays an important role in the induction of inflammation. In this study we demonstrate that the initiation complexes of the lectin pathway, consisting of mannose-binding lectin (MBL) and associated serine proteases (MASPs) elicit Ca2+ signaling in cultured endothelial cells (HUVECs). This is in agreement with our previous results showing that the recombinant catalytic fragment of MASP-1 activates endothelial cells by cleaving protease activated receptor 4. Two other proteases, MASP-2 and MASP-3 are also associated with MBL. Earlier we showed that recombinant catalytic fragment of MASP-2 cannot activate HUVECs, and in this study we demonstrate that the same fragment of MASP-3 has also no effect. We find the same to be the case if we use recombinant forms of the N-terminal parts of MASP-1 and MASP-2 which only contain non-enzymatic domains. Moreover, stable zymogen mutant form of MASP-1 was also ineffective to stimulate endothelial cells, which suggests that in vivo MASP-1 have the ability to activate endothelial cells directly as well as to activate the lectin pathway simultaneously. We show that among the components of the MBL-MASPs complexes only MASP-1 is able to trigger response in HUVECs and the proteolytic activity of MASP-1 is essential. Our results strengthen the view that MASP-1 plays a central role in the early innate immune response

MASP-1 Induces a Unique Cytokine Pattern in Endothelial Cells: A Novel Link between Complement System and Neutrophil Granulocytes

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms

A case report of isolated distal upper extremity weakness due to cerebral metastasis involving the hand knob area

Unilateral weakness of an upper extremity is most frequently caused by traumatic nerve injury or compression neuropathy. In rare cases, lesion of the central nervous system may result in syndromes suggesting peripheral nerve damage by the initial examination. Pseudoperipheral hand palsy is the best known of these, most frequently caused by a small lesion in the contralateral motor cortex of the brain. The 'hand knob' area refers to a circumscribed region in the precentral gyrus of the posterior frontal lobe, the lesion of which leads to isolated weakness of the upper extremity mimicking peripheral nerve damage. The etiology of this rare syndrome is almost exclusively related to an embolic infarction.We present the case of a 70-year-old male patient with isolated left sided upper extremity weakness and clumsiness without sensory disturbance suggesting a lesion of the radial nerve. Nerve conduction studies had normal results excluding peripheral nerve damage. Neuroimaging (cranial CT and MRI) detected 3 space occupying lesions, one of them in the right precentral gyrus. An irregularly shaped tumor was found by CT in the left lung with multiple associated lymph node conglomerates. The metastasis from this mucinous tubular adenocarcinoma with solid anaplastic parts to the 'hand knob' area was responsible for the first clinical sign related to the pulmonary malignancy.Pseudoperipheral palsy of the upper extremity is not necessarily the consequence of an embolic stroke. If nerve conduction studies have normal results, neuroimaging - preferably MRI - should be performed, as lesion in the hand-knob area of the precentral gyrus can also be caused by a malignancy

Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups

IntroductionWhile complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood.MethodsWe therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome.ResultsWe show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID.ConclusionIn conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted

Decreased adhesion to endothelium leads to elevated neutrophil granulocyte count in hereditary angioedema patients

Abstract As many aspects of hereditary angioedema (HAE) due to C1-inhibitor (C1-INH) deficiency (C1-INH-HAE) cannot be explained with elevated bradykinin level alone, it has recently become clear that other factors also play an important role in the pathogenesis. One of these factors could be elevated neutrophil granulocyte (NG) counts, which are associated with increased NG activation in C1-INH-HAE patients; however, their origin has not been elucidated so far. Here, we aimed to investigate whether the excess of NGs is due to disturbed maturation, biased circulating/marginated pool equilibrium or decreased elimination. We enrolled 20 attack-free C1-INH-HAE patients together with 21 healthy controls and collected blood samples. We compared cell surface maturation markers, adhesion molecules, cytokine receptors, and Ca2+-mobilization of NG by flow cytometry, activation markers by ELISA, and NG/endothelial cell adhesion by automated pipetting system. Cell-surface markers showed normal maturation of NGs in C1-INH-HAE patients. Adhesion of NGs to endothelial cells pretreated with lipopolysaccharide or phorbol 12-myristate 13-acetate was significantly weaker in samples from C1-INH-HAE patients and bradykinin had no effect on the adhesion. NGs from C1-INH-HAE patients were in an activated state when assessed by soluble activation markers without any stimulation. Our data support that the maturation of NGs in C1-INH-HAE patients is normal, whereas adhesion properties of patient-derived NGs to the endothelium are reduced compared to those from healthy controls, indicating a bias between the circulating and marginated pools of NGs in patients. Bradykinin may not be responsible for reduced adhesion properties of NGs