COVID-19 cytokine storm: The anger of inflammation.

Patients with COVID-19 who require ICU admission might have the cytokine storm. It is a state of out-of-control release of a variety of inflammatory cytokines. The molecular mechanism of the cytokine storm has not been explored extensively yet. The attachment of SARS-CoV-2 spike glycoprotein with angiotensin-converting enzyme 2 (ACE2), as its cellular receptor, triggers complex molecular events that leads to hyperinflammation. Four molecular axes that may be involved in SARS-CoV-2 driven inflammatory cytokine overproduction are addressed in this work. The virus-mediated down-regulation of ACE2 causes a burst of inflammatory cytokine release through dysregulation of the renin-angiotensin-aldosterone system (ACE/angiotensin II/AT1R axis), attenuation of Mas receptor (ACE2/MasR axis), increased activation of [des-Arg9]-bradykinin (ACE2/bradykinin B1R/DABK axis), and activation of the complement system including C5a and C5b-9 components. The molecular clarification of these axes will elucidate an array of therapeutic strategies to confront the cytokine storm in order to prevent and treat COVID-19 associated acute respiratory distress syndrome.

Complement activation in hidradenitis suppurativa: a new pathway of pathogenesis?

BACKGROUND: Despite the heavy purulence observed in hidradenitis suppurativa (HS), the kinetics of complement anaphylatoxins acting to prime chemotaxis of neutrophils has not been studied. OBJECTIVES: To explore complement activation in HS. METHODS: Circulating concentrations of complement factor C5a, as well as of membrane attack complex C5b-9, were determined in the plasma of 54 treatment-naïve patients and of 14 healthy controls, as well as in the pus of seven patients. Results were correlated with Hurley stage and International Hidradenitis Suppurativa Severity Score. Peripheral blood mononuclear cells (PBMCs) were isolated from seven patients with Hurley stage III HS and seven healthy volunteers and stimulated in the presence of 25% of plasma for the production of tumour necrosis factor-α (TNF-α). RESULTS: Circulating C5a and C5b-9 were significantly greater in patient than in control plasma; however, concentrations in pus were very low. Circulating C5a levels exceeding 28 ng mL-1 were associated with a specificity > 90% with the occurrence of HS. Circulating levels of C5a and C5b-9 were greater in patients with more severe HS. PBMCs of patients produced high concentrations of TNF-α only when growth medium was enriched with patient plasma; this was reversed with the addition of the C5a blocker IFX-1. CONCLUSIONS: Systemic complement activation occurs in HS and may be used as a surrogate biomarker of HS. C5a stimulates overproduction of TNF-α and may be a future therapeutic target.

Brain histopathology in patients with systemic lupus erythematosus: identification of lesions associated with clinical neuropsychiatric lupus syndromes and the role of complement.

OBJECTIVES: Neuropsychiatric (NP) involvement is a poorly understood manifestation of SLE. We studied post-mortem histopathology in relation to clinical NPSLE syndromes and complement deposition in brains of NPSLE and SLE patients and controls. Furthermore, we investigated the correlation between cerebral post-mortem histopathology and ex vivo 7 T MRI findings in SLE and NPSLE. METHODS: A nationwide search for autopsy material yielded brain tissue from 16 NPSLE and 18 SLE patients. Brains obtained from 24 patients who died of acute cardiac events served as controls. Apart from a histopathological evaluation, paraffin-embedded cortical tissue was stained for components of the classical, lectin and terminal complement pathways. RESULTS: Diffuse vasculopathy, microinfarction, macroinfarction, vasculitis and microthrombi occurred significantly more often in NPSLE than SLE patients and were absent in controls. Focal vasculopathy was found in both SLE patients and controls. Complement deposition was strongly associated with both SLE and NPSLE, but not with controls (P < 0.001). Microthrombi were found uniquely in NPSLE and were associated with C4d and C5b-9 deposits (P < 0.05). A 7 T MRI was unable to detect most small vessel injury that was visible histopathologically. CONCLUSION: Our study demonstrates that histopathological lesions in NPSLE represent a continuum, ranging from non-specific lesions such as focal vasculopathy, to more specific lesions including C4d- and C5b-9-associated microthrombi and diffuse vasculopathy related to clinical syndromes defining NPSLE. Complement deposition may be a key factor in the interaction between circulating autoantibodies and thromboischaemic lesions observed in NPSLE. Therefore, complement inhibition may have novel therapeutic potential in NPSLE.

The C5a anaphylatoxin receptor (C5aR1) protects against Listeria monocytogenes infection by inhibiting type 1 IFN expression.

Listeria monocytogenes is a major cause of mortality resulting from food poisoning in the United States. In mice, C5 has been genetically linked to host resistance to listeriosis. Despite this genetic association, it remains poorly understood how C5 and its activation products, C5a and C5b, confer host protection to this Gram-positive intracellular bacterium. In this article, we show in a systemic infection model that the major receptor for C5a, C5aR1, is required for a normal robust host immune response against L. monocytogenes. In comparison with wild-type mice, C5aR1(-/-) mice had reduced survival and increased bacterial burden in their livers and spleens. Infected C5aR1(-/-) mice exhibited a dramatic reduction in all major subsets of splenocytes, which was associated with elevated caspase-3 activity and increased TUNEL staining. Because type 1 IFN has been reported to impede the host response to L. monocytogenes through the promotion of splenocyte death, we examined the effect of C5aR1 on type 1 IFN expression in vivo. Indeed, serum levels of IFN-α and IFN-ß were significantly elevated in L. monocytogenes-infected C5aR1(-/-) mice. Similarly, the expression of TRAIL, a type 1 IFN target gene and a proapoptotic factor, was elevated in NK cells isolated from infected C5aR1(-/-) mice. Treatment of C5aR1(-/-) mice with a type 1 IFNR blocking Ab resulted in near-complete rescue of L. monocytogenes-induced mortality. Thus, these findings reveal a critical role for C5aR1 in host defense against L. monocytogenes through the suppression of type 1 IFN expression.

Inhibition of the membrane attack complex of the complement system reduces secondary neuroaxonal loss and promotes neurologic recovery after traumatic brain injury in mice.

Traumatic brain injury (TBI) is the leading cause of disability and death in young adults. The secondary neuroinflammation and neuronal damage that follows the primary mechanical injury is an important cause of disability in affected people. The membrane attack complex (MAC) of the complement system is detected in the traumatized brain early after TBI; however, its role in the pathology and neurologic outcome of TBI has not yet been investigated. We generated a C6 antisense oligonucleotide that blocks MAC formation by inhibiting C6, and we compared its therapeutic effect to that of Ornithodoros moubata complement inhibitor (OmCI), a known inhibitor of C5 activation that blocks generation of the anaphylatoxin C5a and C5b, an essential component of MAC. Severe closed head injury in mice induced abundant MAC deposition in the brain. Treatment with C6 antisense reduced C6 synthesis (85%) and serum levels (90%), and inhibited MAC deposition in the injured brain (91-96%). Treatment also reduced accumulation of microglia/macrophages (50-88%), neuronal apoptosis, axonal loss and weight loss (54-93%), and enhanced neurologic performance (84-92%) compared with placebo-treated controls after injury. These data provide the first evidence, to our knowledge, that inhibition of MAC formation in otherwise complement-sufficient animals reduces neuropathology and promotes neurologic recovery after TBI. Given the importance of maintaining a functional complement opsonization system to fight infections, a critical complication in TBI patients, inhibition of the MAC should be considered to reduce posttraumatic neurologic damage. This work identifies a novel therapeutic target for TBI and will guide the development of new therapy for patients.

Differential timing of antibody-mediated phagocytosis and cell-free killing of invasive African Salmonella allows immune evasion.

Nontyphoidal Salmonellae commonly cause fatal bacteraemia in African children lacking anti-Salmonella antibodies. These are facultative intracellular bacteria capable of cell-free and intracellular survival within macrophages. To better understand the relationship between extracellular and intracellular infection in blood and general mechanisms of Ab-related protection against Salmonella, we used human blood and sera to measure kinetics of Ab and complement deposition, serum-mediated bactericidal killing and phagocytosis of invasive African Salmonella enterica serovar Typhimurium D23580. Binding of antibodies peaked by 30 s, but C3 deposition lagged behind, peaking after 2-4 min. C5b-9 deposition was undetectable until between 2 and 6 min and peaked after 10 min, after which time an increase in serum-mediated killing occurred. In contrast, intracellular, opsonized Salmonellae were readily detectable within 5 min. By 10 min, around half of monocytes and most neutrophils contained bacteria. The same kinetics of serum-mediated killing and phagocytosis were observed with S. enterica Typhimurium laboratory strain SL1344, and the S. enterica Enteritidis African invasive isolate D24954 and laboratory strain PT4. The differential kinetics between cell-free killing and phagocytosis of invasive nontyphoidal Salmonella allows these bacteria to escape the blood and establish intracellular infection before they are killed by the membrane attack complex.

[Polymyositis revealing a Sjogren's syndrome]. / Polymyosite révélant un syndrome de Gougerot-Sjögren.

INTRODUCTION: Near 10 to 20% of patients with myositis have another systemic, sometimes inaugural, disease. CASE REPORT: A 48-year-old woman was admitted with progressive hypoesthesia in V2 and V3 areas on both sides, difficulties to chew and swallow and then, proximal and axial muscular deficiency, with weight loss. Brain MRI showed gadolinium-enhanced trigeminal nerves and biological tests revealed anti-SSA and anti-Pm/Scl antibodies and a grade IV in Chisholm scoring system on the labial salivary gland biopsy. Neurophysiological studies revealed a myogenic pattern on tibialis anterior muscles and a muscle biopsy confirmed the diagnosis of polymyositis. CONCLUSION: The diagnosis of primitive Sjogren's syndrome was suspected because of the association of bilateral trigeminal neuropathy and anti-SSA and anti-Pm/Scl antibodies.

The deposition of C5b-9 complexes and its precursors on E. coli J5 during complement activation enhances uptake and toxicities of gentamicin.

The complement system provides the host with protection against pathogenic agents and in some cases can result in damage to host tissue. However, the exact mechanism of how complement kills gram-negative bacteria in lysozyme-neutralized and or lysozyme-depleted serum is still under active investigation. In previous studies, it has been demonstrated that inner membrane damage by the membrane attack complex contributes to depolarization and the subsequent collapse of the membrane potential. In these studies we have shown that the membrane attack complex and its precursors provide additional protective effect by the enhanced uptake of antibiotics in the death of E. coli J5. Specifically, the deposition of C5b fragments from C6 neutralized Pooled Normal Human Serum (PNHS) and C5b6 complexes from C7 neutralized PNHS on E. coli J5 contribute to antibiotic uptake and killing. Since C5b and C5b6 do not form pores, we suggest that disturbances and or cracks in the outer membrane by the deposited complexes accelerates uptake of the antibiotics and enhanced killing of E. coli J5 employed in these studies.

Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses.

The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab.

Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice.

Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC's regulator CD59. However, there is no available C9 deficient mice (mC9(-/-)) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9(-/-). We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1ß in mC9(-/-), which is associated with suppression of MAC-mediated inflammasome activation in mC9(-/-). Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation.

Antibodies reactive to cleaved sites in complement proteins enable highly specific measurement of soluble markers of complement activation.

An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.

Quantification of complement C5b-9 binding to cells by flow cytometry.

Interaction of the complement system, directly or indirectly (e.g., via antibodies), with cells activates the early and late complement components and culminates in the deposition of a membrane-spanning C5b-9 complex on the cell surface. At a high copy number, this C5b-9 will activate cell death, whereas at a low copy number, it will transmit various signals into cells. Quantification of C5b-9 deposition is useful for assessments of the capacity of cells and antibodies to activate complement. By using an antibody that identifies a novel antigen of the C5b-9 complex, the amount of C5b-9 complexes on cells can be quantified by flow cytometry. The detailed protocol is described in this chapter.

Proteomic profiling of secreted proteins for the hematopoietic support of interleukin-stimulated human umbilical vein endothelial cells.

Human umbilical cord vein endothelial cells (HUVECs) secrete a number of factors that greatly impact the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). These factors remain largely unknown. Here, we report on the most comprehensive proteomic profiling of the HUVEC secretome and identified 827 different secreted proteins. Two hundred and thirty-one proteins were found in all conditions, whereas 369 proteins were identified only under proinflammatory conditions following IL-1ß, IL-3, and IL-6 stimulation. Thirteen proteins including complement factor b (CFb) were identified only under IL-1ß and IL-3 conditions and may potentially represent HSPC proliferation factors. The combination of bioinformatics and gene ontology annotations indicates the role of the complement system and its activation. Furthermore, CFb was found to be transcriptionally strongly upregulated. Addition of complement component 5b-9 (C5b-9) monoclonal antibody to the stem cell expansion assay was capable of significantly reducing their proliferation. This study suggests a complement-mediated cross-talk between endothelial cells and HSPCs under proinflammatory conditions.

Complement activation in experimental and human temporal lobe epilepsy.

We investigated the involvement of the complement cascade during epileptogenesis in a rat model of temporal lobe epilepsy (TLE), and in the chronic epileptic phase in both experimental as well as human TLE. Previous rat gene expression analysis using microarrays indicated prominent activation of the classical complement pathway which peaked at 1 week after SE in CA3 and entorhinal cortex. Increased expression of C1q, C3 and C4 was confirmed in CA3 tissue using quantitative PCR at 1 day, 1 week and 3-4 months after status epilepticus (SE). Upregulation of C1q and C3d protein expression was confirmed mainly to be present in microglia and in a few hippocampal neurons. In human TLE with hippocampal sclerosis, astroglial, microglial and neuronal (5/8 cases) expression of C1q, C3c and C3d was observed particularly within regions where neuronal cell loss occurs. The membrane attack protein complex (C5b-C9) was predominantly detected in activated microglial cells. The persistence of complement activation could contribute to a sustained inflammatory response and could destabilize neuronal networks involved.

New insights into the pathogenesis of membranous glomerulonephritis.

PURPOSE OF REVIEW: Membranous nephropathy is one of the most common glomerulopathies. Current treatments are entirely empirical, and concept-driven therapies are dramatically lacking. This review focuses on new pathophysiologic aspects of the disease, with special emphasis on the antigenic targets of pathogenic antibodies. RECENT FINDINGS: Neutral endopeptidase - a podocyte antigen that can digest biologically active peptides - was recently identified as the target antigen of antibodies deposited in the subepithelial space of glomeruli in a subset of patients with antenatal membranous nephropathy. The mothers became immunized because they are deficient in neutral endopeptidase due to truncating mutations in the gene. Membranous nephropathy could be transferred to the rabbit by injection of mothers' immunoglobulin. Development of the renal disease was associated with anti-neutral endopeptidase IgG1. SUMMARY: Membranous nephropathy most likely is a heterogeneous disease, although a common denominator may be that podocytes provide antigenic targets for in-situ formation of glomerular immune deposits. Identification of neutral endopeptidase and additional (podocyte) antigens and characterization of their epitopes should make it possible to design more effective and better tolerated therapies. Fetomaternal alloimmunization is a novel mechanism of renal disease that may apply to other organs as well.