RBC Adhesive Capacity Is Essential for Efficient 'Immune Adherence Clearance' and Provide a Generic Target to Deplete Pathogens from Septic Patients
Authors: De Back Djuna Zoe , Kostova Elena , Klei Thomas , Beuger Boukje , van Zwieten Rob , Kuijpers Taco , Juffermans Nicole , van den Berg Timo , Korte Dirk , van Kraaij Marian , van Bruggen Robin 
Published: November 2019

Abstract
Sepsis is a life threatening organ dysfunction caused by a dysregulated host response to infection. It is a global health care problem and the leading cause of death in ICU patients. A rather unknown but effective mechanism to protect ourselves from such pathogen invasion is immune-adherence clearance (IAC). During IAC pathogens (including bacteria, fungi and viruses) can bind to red blood cells (RBCs) through complement opsonization and subsequent binding to complement receptor 1 (CR1, CD35) expressed on their cell surface. After binding of the pathogen to CR1, the RBCs deliver the pathogens to macrophages of the spleen and liver, where they are phagocytosed and degraded.
To study the underlying mechanisms of IAC in more detail, we have developed an assay to monitor the transfer of opsonized pathogens bound to RBCs to human monocytes and/or macrophages under flow conditions, using confocal microscopy. The transfer of a variety of different pathogens, including S. aureus, E. coli and C. albicans, was studied in this assay. When looking at the transfer process in detail, it was noticed that RBC attach to the phagocyte prior to the transfer and remain attached shortly after the transfer, suggesting that RBC might be interacting with phagocytes through adhesion molecules such as integrins to establish firm binding between these two cell types. To test this hypothesis several adhesion molecules, on the phagocytes as well as on the RBC, were blocked by monoclonal antibodies and IAC was quantified. First, complement receptor 3 (CR3, amb2 CD11b/CD18 integrin) an important receptor for adhesion, migration and phagocytosis was blocked by monoclonal antibodies on the phagocytes, after which bacterial transfer was impaired. This indicated that CR3 is crucial for efficient IAC. To confirm the involvement of CR3 in IAC, we analyzed the monocytes of a known LAD-1 patient. This rare immunodeficiency is characterized by an inherited molecular defect of the β2integrin subunit (CD18) which results in impaired adhesion and migration of the patient's leukocytes and clinically manifests in recurrent infections. Our results showed a highly reduced interaction between the phagocytes and the RBCs, resulting in a decrease in bacterial transfer of >70%.
Next, we tested the effects of blocking antibodies directed against several RBC adhesion molecules on the transfer process. When blocking CD147 (Basigin, Ok blood group) and ICAM-4 (CD242, Landsteiner-Wiener), but not Glycophorin A (GPA, CD235a) on the RBC the transfer process was largely inhibited. These findings demonstrate the involvement of direct cell-cell interactions between RBC and macrophages in IAC and provide evidence that RBC adopt a "sticky" phenotype after binding a pathogen through CR1, which enables phagocytes to bind them under flow. We anticipated that blood bank filters (currently used for leukocyte depletion) might be used to bind and filter RBC-pathogen complexes from blood due to their "sticky" phenotype. This was found to be possible in a series of experiments, not only using in vitrogenerated RBC-pathogen complexes, but also using blood from septic patients. Filtration using a standard leukocyte reduction filter showed reduction of RBC-pathogen complexes close to 100%, independent of the type of pathogen. We foresee that this knowledge can be used to develop a generic approach to deplete RBCs carrying pathogens specifically from the blood stream, and thereby filter RBC-pathogen complexes from the blood of patients suffering from sepsis.

Disclosures
No relevant conflicts of interest to declare.