Session 11 - Complement deficiency in Immunopathology

Chairs: Veronique Fremeaux-Bacchi Santiago & Rodriguez de Cordoba

Background: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59, leading to loss of function, have been described in 18 patients. Here we investigated single cell RNA sequencing of PBMCs from a patient deficient in CD59.

Methods: Single Cell RNA Sequencing. PBMCs from a patient with CD59 homozygous mutation and an age- and gender-matched control were diluted with FACS buffer to a concentration of 0.6–0.7×106 cells/ml and viability above 90% was confirmed. RNA from the barcoded cells from each sample was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a chromium single cell 3′ v2 reagent kit (10× Genomics) according to the manufacturer’s instructions. Sequencing was performed with the Illumina NextSeq 500 high-output kit (Illumina, San Diego, CA, USA) for 75 cycles with 26bp read from read1 and 58bp from read2. Single-Cell RNA-Seq Analysis. Single-cell RNA-seq analyses were performed used Seurat 4.3.0. Uniform Manifold Approximation and Projection (UMAP) was then performed. Clusters were determined using the shared nearest neighbor (SNN) modularity optimization-based clustering algorithm. Differentially expressed markers were identified.

Results: PBMCs from the patient with homozygous CD59 deficiency showed marked depletion of NK cells (36% of normal) and marked elevation in CD4:CD8 ratio; 6.14 compared to 3.75 in normal control. Tregs were twice in number in the patient and both CD4 and CD8 cells expressed differentially gene clusters. B cells were a little bit elevated in the patient, but naïve B cells were elevated 2.5 folds. In one cluster SNHG5, RPS28, ARL4C, HLA-DPB1, AES, ABRACL, CSTB, BAX, CAP1, were elevated along with others that were differentially negatively expressed. Monocytes were relatively depleted in the patient. These gene changes fitted to known patterns in antigen presentation, TH17 signaling pathway, type I diabetes and other autoimmunity, GvHD, and some viral and bacterial infections.

Conclusion: SCA reveals expected and unexpected changes in PBMC subpopulations from membrane CD59 deficient patient. These finding may elucidate unknown functions of both canonic and non-canonic CD59 functions.

Dror Mevorach

Background: C3 glomerulopathy (C3G) is a rare kidney disorder associated with complement alternative pathway (AP) dysregulation and C3 deposition within the glomerulus.

Proteinuria and nephrotic syndrome are present at onset in 90% and 30% of C3G patients, respectively. A morphological hallmark of nephrotic syndrome is effacement of foot processes in podocytes, highly specialized cells that play a main role in glomerular permeability.

Through exome sequencing, we identified in a proband from a trio with C3G and nephrotic syndrome a variant in a new candidate gene, IQGAP1, encoding for the IQ Motif Containing GTPase Activating Protein 1 (IQGAP1). IQGAP1 is a scaffold protein largely expressed in the podocytes, where it interacts with proteins of the slit-diaphragm complex and cytoskeleton, regulating podocytes morphology, motility and permeability. The novel de novo heterozygous 1-base pair deletion leads to a frameshift with the introduction of a premature stop codon (p.L159Rfs*4).

This preliminary finding led us to investigate the link between IQGAP1 alterations and complement activation in podocytes.

Methods: Conditionally immortalized human podocytes were transfected with null or IQGAP1 small interfering RNA (siRNA). IQGAP1 mRNA expression levels were evaluated by real-time PCR, and IQGAP1 and cytoskeleton proteins localization by immunofluorescence. C3 deposition was evaluated, using confocal microscopy, by incubating transfected podocytes with 10% normal human serum (NHS).

Results: Transfection of podocytes with IQGAP1 siRNA, which recapitulates the effect of the frameshift identified in C3G patient, induced a decrease of IQGAP1 mRNA expression by 80% and caused a reorganization of podocyte cytoskeleton proteins, such as integrin-linked kinase (ILK) and podocin, and a cortical accumulation of F-actin. Incubation of siRNA transfected podocytes with NHS induced a three-fold increase in C3 deposition as compared to podocytes transfected with null siRNA (n=5; IQGAP siRNA: 1259 pixels2; null siRNA:377 pixels2; p=0.01). C3 deposits were abrogated by sCR1.

Conclusions: Altogether, these findings support the hypothesis that intrinsic defects altering the podocyte structure and/or function may predispose to a secondary complement attack on the glomerular cells, contributing to C3G onset. Further studies are needed to validate this hypothesis and to shed light on the molecular players involved in C3G.

Lisa Quadri

Background: Ficolin-3 is the main initiator of the lectin pathway in humans. Case reports of ficolin-3 deficient patients have suggested that ficolin-3 deficiency may be enriched in patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease where complement plays an important role. Therefore, this study aimed to investigate the activity levels of ficolin-3 and to identify potential ficolin-3 deficient individuals in two Swedish SLE cohorts.

Methods: Serum or plasma samples from SLE patients (n=810) and matched controls (n=566) were collected from the Karolinska Institute (KI) and Umeå University Hospital. The ficolin-3 activity levels were measured by an in-house developed functional ELISA with a pooled normal human serum sample as a reference. Serial samples were analyzed for ficolin-3 deficient patients when available. Sequencing data were analyzed for FCN3 frame-shift mutation +1637delC (rs532781899) and other potential loss-of-function (LoF) variants.

Results: This screening revealed that the level of ficolin-3 activity varies largely in patients with SLE. The activity levels also show that SLE patients seem to generally have elevated ficolin-3 activity compared to the control group (p<0.0001). Out of 810 patients with SLE, four patients were determined to be ficolin-3 deficient. For two of these patients, the ficolin-3 activity was at normal levels at the time of diagnosis and thereafter depleted over time, indicating an acquired deficiency. For deficient patients, no or very low ficolin-3 protein levels and no lectin pathway-dependent complement activation could be detected. Autoantibodies against ficolin-3 were not detectable. No patients were homozygous for the +1637delC frameshift mutation, whereas in total 10 patients were determined to be heterozygous carriers. These heterozygous patients displayed lower levels of ficolin-3 activity but did not include the deficient patients. Additional possible LoF variants were analyzed but none were enriched in either patients or controls.

Conclusions: Contrary to the classical pathway of the complement system we show that genetic ficolin-3 deficiency is not a risk factor for SLE. Instead, acquired ficolin-3 deficiency was observed in a subgroup of SLE patients, possibly due to a potent activation of the lectin pathway that depleted ficolin-3 plasma levels in these individuals.

Linnea Lindelof

C3 is the most abundant protein of the Complement System and plays an important role in the immune response. C3-deficient patients are more susceptible to recurrent and severe infection diseases. Several studies have demonstrated the importance of C3 in controlling infection, but its role in leukocyte differentiation and cellular response is still poorly understood. This study aimed to evaluate several cellular parameters in bone marrow differentiated macrophages (BMDM) and thioglycollated-elicited peritoneal macrophages (TEPM) from C57Black/6 C3-/- (B6.C3-/-; C3KO) and C57Black/6 (B6.C3+/+; wild type) mice. The total number of cells in the peritoneal exudate was not affected by the absence of C3. TEPM F4/80high cells, resident macrophages that do not depend on the differentiation of new circulating monocytes, are present at higher levels in B6.C3-/- mice than in B6.C3+/+ mice. The morphology of the macrophage cytoskeleton plays a crucial role in migration, adhesion and engulfment of particles. We observed an increase in cell roundness and more solid morphology (fewer invaginations) in TEPM B6.C3-/- animals, which may suggest that these cells would be less activated. We did not observe the same phenomenon in BMDM. Microtubules are essential for cell displacement into the inflammatory site. In this study, we observed that in the absence of C3, the presence of F-tubulin filaments increases in both BMDM and TEPM. In addition, a significant increase in CR4 (CD11c/CD18) and CD64 (FcgammaRI) expression was detected in both BMDM and TEPM from B6.C3-/- mice compared to the same cells from B6.C3+/+ mice. ROS production and MAPK activation after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) was lower in B6.C3-/- macrophages than their wild type counterparts, suggesting the importance of C3 for cell signaling. As expected, BMDM and TEPM from B6.C3-/- mice phagocytosed less serum-opsonized zymosan particles than the corresponding cells from B6.C3+/+ mice. Together our results suggest that C3 plays a role in macrophage phenotype and activation.

Lourdes Isaac

Morning coffee


Plenary 3: Muzz Haniffa - Immune Cell Regulation of Skin Morphogenesis

Chair: Claire Harris

Session 12 - Translational models of disease

Chairs: Elena Goicoechea de Jorge & Robert Reiben

Excessive activation of complement, a crucial component of innate immunity, causes many diseases such as paroxysmal nocturnal hemoglobinuria (PNH) and age-related macular degeneration (AMD). Among the complement components, C3 is central for all complement activation pathways, thus an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for treating PNH and dry AMD. However, most, if not all of these C3 inhibitors are primate C3-specific, making evaluating their treatment efficacies in vivo before a clinical trial extremely difficult and costly. We previously developed a C3 knockout (KO) rat. By supplementing purified human C3 protein into the blood of the C3 KO rats, we found that human C3 is compatible to the rat complement system. We thus developed a human C3 knockin (KI) rat by knocking in a human C3 expression construct into the rat C3 loci using CRISPR/Cas 9 technology. The resultant human C3 KI rats expressed human but not rat C3 as examined by respective ELISA, Western blot and/or RT-PCR, and possessed complement activities both in vitro and in vivo. More importantly, the complement hemolytic activity of the human C3 KI rats was inhibited by compstatin, a C3 inhibitor known to be specific only to primate C3. The successful development of human C3 KI rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.

Feng Lin

Background: Complement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin.

Methods: Tail bleeding time and hemolysis were measured in FH-deficient mice (CFH-/-). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR).

Results: Tail bleeding time in CFH-/- mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed. Neither FH complement regulatory function nor complement haemolytic activity was altered by thrombin binding.

Conclusion: Complement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis.

Meike Heurich

Background: Polytrauma as a life threatening multiple injury leads to rapid activation of the cross-talking coagulation- and complement systems. However, excessive complement activation can contribute to systemic inflammation and organ dysfunction. In the case of sepsis, blockade of central complement components and TLR molecules appeared to be beneficial as we also documented recently in a pig model with 72 hrs observation using C5 and CD14 inhibition. Therefore, we hypothesized that absence of the central complement component C3 and the TLR 4-coreceptor CD14 will improve cellular and organ function post trauma in genetically modified mice.

Methods: Polytrauma plus hemorrhagic shock (HS) was modelled in C57bl/6 mice by blunt traumatic brain injury, thoracic trauma, femur fracture and soft tissue injury. After the trauma impact, animals were hemodynamically monitored in our mouse intensive-care unit. Sham procedures and polytrauma/HS was applied in genetic absence or presence of C3, or CD14 or both, C3/CD14 (n=8/group). As read-outs, we investigated the hemodynamic and inflammatory response aas well as multi-organ function 4 h after injury. Statistical analyses were performed by ANOVA with post hoc testing.

Results: The hemodynamic changes after the polytrauma/HS did not significantly alter between the C3/CD14 sufficient and deficient mice. Systemic inflammation assessed by IL-6 plasma concentrations, multi-organ performance measured by the established biomarkers CC16, IFABP, GFABP, NGAL, among others, and clinical outcome (reflected by mortality rate) did not significantly alter between the C3 deficient, CD14 deficient and C3/CD14 double knock out mice in the early observation period up to 4 h post polytrauma/HS. Of note, the polytrauma-induced increase in the endothelial damage marker syndecan-1 was significantly reduced in C3-/- mice.

Conclusion: Whereas long-term effects after severe tissue trauma seem to benefit from blockade of complement (C5) and TLRs (CD14) , our current data in mice suggest, that very early after polytrauma/HS neither complement nor TLR inhibition alter the inflammatory and organ response, except for the endothelial damage. These differential-temporal findings indicate that the therapeutic effect of this inhibition is not observed early, but might have a major impact at a later stage.

Markus Huber-Lang

Background: Traumatic brain injury (TBI) gives rise to a secondary neuroinflammation that can last years after injury and is associated with the development of progressive cognitive impairment and chronic disabilities. We and others have shown that activation of an immune response, including activation of complement, plays a central role in this process by mediating the aberrant phagocytosis of perilesional neurons and synapses. However, since the immune system can promote both brain repair and brain damage, identifying the cellular components and their temporal distribution would contribute to our understanding of the neuroinflammatory process and help identify TBI treatment strategies.

Methods: Using a controlled cortical impact TBI model, we leveraged mass cytometry to evaluate how complement and complement inhibition affects the immune landscape after TBI. We designed a 35-antibody panel to identify resident and peripheral immune cells and their functional receptors involved in phagocytosis, chemotaxis, and complement signaling at various time points after TBI. We used the injury-site targeted complement inhibitor, CR2-Crry, consisting of the complement inhibitor (Crry) linked to a fragment of complement receptor 2 (CR2), which increases complement inhibition bioavailability and efficacy without exerting systemic immunosuppressive effects.

Results: We found an increase in the percentage of infiltrating peripheral immune cells at acute time points and identified distinct microglia sub-populations, among which one subpopulation expressing complement receptor 4 (CR4) showed progressive increase through at least 28 days after injury. CR2-Crry treatment after TBI improved cognitive functions, impacted the expression of functional receptors on infiltrating cells, and reduced the abundance of brain resident immune cells. Conversely, inhibition of C5aR effectively reduced the infiltration of immune cells, but did not improve histological or behavioral outcomes, which suggests the neuroprotective effect of CR2-Crry is not mediated via inhibition of C5a generation.

Conclusions: CR2-Crry improved post-TBI outcomes and altered immune cell infiltration, as well as complement receptor and phagocytic receptor expression. Together with data from previous studies and the current C5aR inhibition data, the results indicate that the neuroprotective effect of CR2-Crry is likely via modulating C3 opsonization and complement receptor expression.

Silvia Guglietta

Background: Mannan-binding lectin-associated serine protease 3 (MASP3) converts pro-factor D (pro-FD) to mature FD. It was reported earlier that only mature FD was catalytically active, making MASP3 a key component of the alternative pathway (AP) complement and an attractive drug target. However, the role of MASP3 in AP complement has been controversial as various levels of AP activity were detected in the absence of MASP3, both in human and in mice. We have recently demonstrated significant AP complement activity in a newly generated Masp3-/- mouse strain at high (20-50%) but not low (5-10%) plasma concentrations and attributed this activity to intrinsic catalytic activity of pro-FD (1). The aim of this study is to assess the functional significance of this partial activity in AP complement-mediated tissue injury.

Methods: We crossed the Masp3-/- mouse with a factor H mutant mouse (FHR/R) that develops AP complement-dependent atypical hemolytic uremic syndrome and systemic thrombophilia (2) and assessed the effect of MASP3 deficiency on disease development by evaluating FHR/R and FHR/RMasp3-/- littermates. 

Results: We found that MASP3 deficiency significantly reduced mortality, and ameliorated thrombocytopenia, anemia, and renal injury in FHR/R mice. It also prevented systemic thrombophilia and macro-vessel thrombosis. However, about 30% of FHR/RMasp3-/- mice (5/17) still died prematurely because of extra-renal organ injury including intestinal and pulmonary hemorrhage and cerebral ischemia. These findings contrasted with the phenotype of other AP complement and FHR/R double knockout mice such as FHR/RC3-/- or FHR/RFD-/- mice which showed complete disease rescue. AP complement activity in FHR/RMASP3-/- mice, as measured in 20% serum using an LPS-based ELISA assay, was significantly lower than that of FHR/Rmice but significantly higher than that of FHR/RFD-/- mice.

Conclusion: Partial AP complement activity afforded by pro-FD in the absence of MASP3-/- was sufficient to cause tissue injury and therapeutic targeting of MASP3 may not be completely efficacious in all settings of AP complement-mediated diseases.

1. Gullipalli D, Miwa T, Golla M, Sato S, Angampalli S, Song WC. J Immunol. 2023 Mar 29:ji2200932. doi: 10.4049/jimmunol.2200932.
2. Ueda Y et al Blood. 2017 Mar 2;129(9):1184-1196.

Takashi Miwa

Complement as an essential part of innate immunity maintains immune homeostasis and plays a key role in several biological processes, such as removal of injured cells and debris, host defense injuries and repair. However, excessive complement activation can lead to diseases. Therefore, this cascade needs to be tightly regulated. Factor H-related protein 1 (FHR1) is a member of Factor H protein family involved in complement control and in the regulation of the innate immune response. In humans, homozygous deletion of FHR1 and FHR3 genes (∆∆FHR3/FHR1) has been reported to be protective against diseases such as immunoglobulin A nephropathy (IgAN) and age-related macular degeneration (AMD), but has also been shown as a risk for atypical hemolytic-uremic syndrome (aHUS) and atherosclerotic cardiovascular diseases (ACVD) (Skerka et al., 2021; Irmscher et al., 2021). Recently, our group also revealed that FHR1 is an inflammatory mediator inducing proinflammatory response of monocytes (Irmscher et al., 2021). Currently, the role of FHR1 has not been fully explored in animal models. Here, we generated a FHR-E-/- (FHR1 murine homolog) knock-out mouse model for investigating further the role of FHR-E in vivo. We first measured plasma levels of complement C3 and the regulator Factor H in FHRE-/- and WT mice. We observed related levels in both type of animals. In addition expression of pro- and anti-inflammatory cytokines in FHR-E-/- and in WT mice was evaluated in several tissues including kidney, liver and heart. Our data revealed that in most of these tissues, cytokines expression such as IL-1β and TNFα are significantly reduced both at RNA and protein levels in FHR-E-/- mice as compared to WT animals. Further analyses also revealed altered levels of reactive oxygen species in FHRE-/- mice. Taken together, the present study suggests that deletion of the FHR-E gene is more protective against inflammation, confirming our previous observations made in humans. Since this study is conducted in vivo, it provides real insights into the mechanisms how FHR-E regulates the complement system and inflammation and lays the groundwork for further studies.

Tomas Ghebreslassie


13.15 - 18.00

Social Excursions

19.00 - 00.00

Conference Dinner

Please note that a Dinner ticket needed to be booked in advance. If you aren’t sure if you are booked to attend  the dinner, please email info@icw2023newcastle.co.uk

It is a 7pm arrival and drinks with dinner to start at 7.30pm. We have a fantastic Ceilidh band organised for you during the evening. A Ceilidh Band is a traditional folk band with a caller that will walk you through a series of dance moves!

Please wear or bring your conference badge along with you.

The dinner will take place at:

Newcastle Civic Centre, Banqueting Hall, Barras Bridge ,Newcastle upon Tyne, NE1 8QH

Map here

Dress Code: smart/casual (we recommend wearing comfortable shoes to join in with the Ceilidh)