Elevated expression of complement factor I in lung cancer cells associates with shorter survival-Potentially via non-canonical mechanism.

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

Complement activation drives antibody-mediated transfusion-related acute lung injury via macrophage trafficking and formation of NETs.

ABSTRACT: Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and, to date, is without available therapies. Here, we investigated the role of the complement system in TRALI. Murine anti-major histocompatibility complex class I antibodies were used in TRALI mouse models, in combination with analyses of plasma samples from patients with TRALI. We found that in vitro complement activation was related to in vivo antibody-mediated TRALI induction, which was correlated with increased macrophage trafficking from the lungs to the blood in a fragment crystallizable region (Fc)-dependent manner and that this was dependent on C5. Human immunoglobulin G 1 variants of the murine TRALI-inducing antibody 34-1-2S, either unable to activate complement and/or bind to Fcγ receptors (FcγRs), revealed an essential role for the complement system, but not for FcγRs, in the onset of 34-1-2S-mediated TRALI in mice. In addition, we found high levels of complement activation in the plasma of patients with TRALI (n = 53), which correlated with elevated neutrophil extracellular trap (NET) markers. In vitro we found that NETs could be formed in a murine, 2-hit model, mimicking TRALI with lipopolysaccharide and C5a stimulation. Collectively, this reveals a critical role of Fc-mediated complement activation in TRALI, with a direct relation to macrophage trafficking from the lungs to the blood and an association with NET formation, suggesting that targeting the complement system may be an attractive therapeutic approach for combating TRALI.

Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

Serum neurofilament light chain, contactin-1 and complement activation in anti-MAG IgM paraprotein-related peripheral neuropathy.

INTRODUCTION: In anti-myelin-associated glycoprotein IgM paraprotein-related peripheral neuropathy (anti-MAG PN), there is a lack of reliable biomarkers to select patients eligible for therapy and for evaluating treatment effects, both in routine practice and in clinical trials. Neurofilament light chain (NfL) and contactin-1 (CNTN1) can serve as markers of axonal and paranodal damage. Complement activation is involved in the pathogenesis in anti-MAG PN. We, therefore, hypothesized that serum NfL, CNTN1, C3b/c and C4b/c may function as biomarkers of disease activity in anti-MAG PN. METHODS: In this prospective cohort study, we included 24 treatment-naïve patients with anti-MAG PN (mean age 69 years, 57% male) that had IgM paraproteinemia, a high IgM MAG-antibody, and clinical diagnosis of anti-MAG PN by a neurologist specialized in peripheral nerve disorders. We measured serum NfL, CNTN1, C3b/c and C4b/c, reference values were based on healthy controls. As controls, 10 treatment-naïve patients with IgM Monoclonal gammopathy of undetermined significance (MGUS) or Waldenström's Macroglobulinemia (mean age 69 years, 60% male) without signs of neuropathy were included (non-PN). RESULTS: NfL, CNTN1 levels in serum were mostly normal in anti-MAG PN patients and comparable to non-PN patients. C3b/c and C4b/c levels were normal in anti-MAG PN patients. CONCLUSION: Our results do not support serum NfL, CNTN1, and C3b/c and C4b/c as potential biomarkers in anti-MAG PN, although we cannot exclude that subgroups or subtle abnormalities could be found in a much larger cohort with longitudinal follow-up.

S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation.

Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.

Therapeutic Lessons to be Learned From the Role of Complement Regulators as Double-Edged Sword in Health and Disease.

The complement system is an important part of the innate immune system, providing a strong defense against pathogens and removing apoptotic cells and immune complexes. Due to its strength, it is important that healthy human cells are protected against damage induced by the complement system. To be protected from complement, each cell type relies on a specific combination of both soluble and membrane-bound regulators. Their importance is indicated by the amount of pathologies associated with abnormalities in these complement regulators. Here, we will discuss the current knowledge on complement regulatory protein polymorphisms and expression levels together with their link to disease. These diseases often result in red blood cell destruction or occur in the eye, kidney or brain, which are tissues known for aberrant complement activity or regulation. In addition, complement regulators have also been associated with different types of cancer, although their mechanisms here have not been elucidated yet. In most of these pathologies, treatments are limited and do not prevent the complement system from attacking host cells, but rather fight the consequences of the complement-mediated damage, using for example blood transfusions in anemic patients. Currently only few drugs targeting the complement system are used in the clinic. With further demand for therapeutics rising linked to the wide range of complement-mediated disease we should broaden our horizon towards treatments that can actually protect the host cells against complement. Here, we will discuss the latest insights on how complement regulators can benefit therapeutics. Such therapeutics are currently being developed extensively, and can be categorized into full-length complement regulators, engineered complement system regulators and antibodies targeting complement regulators. In conclusion, this review provides an overview of the complement regulatory proteins and their links to disease, together with their potential in the development of novel therapeutics.