Immunomodulatory Roles of Red Blood Cells

Abstract

Purpose of the Review:

To discuss recent advances supporting the role of Red Blood Cells in the host immune response

Recent Findings:

Over the last century, research has demonstrated that red blood cells exhibit functions beyond oxygen transport, including immune function. Recent work indicates that the nucleic acid sensing receptor, Toll-like Receptor 9 (TLR9), is expressed on the RBC surface and implicated in innate immune activation and red cell clearance during inflammatory states. In addition to this DNA-sensing role of RBCs, there is growing evidence that RBCs may influence immune function by inducing vascular dysfunction. RBC proteomics and metabolomics have provided additional insight into RBC immune function, with several studies indicating changes to RBC membrane structure and metabolism in response to SARS-CoV-2 infection. These structural RBC changes may even provide insight into the pathophysiology of the “long-COVID” phenomenon. Finally, evidence suggests that RBCs may influence host immune responses via complement regulation. Taken together, these recent findings indicate RBCs possess immune function. Further studies will be required to elucidate better how RBC immune function contributes to the heterogeneous host response during inflammatory states.

Summary:

The appreciation for non-gas exchanging, red blood cell immune functions is rapidly growing. A better understanding of these RBC functions may provide insight into the heterogeneity observed in the host immune response to infection and inflammation.

Introduction

Red blood cells (RBCs) are the most abundant host cell in the human body, outnumbering the following most abundant circulating blood cells by several hundred times.[1] While overlooked in immunology as inert, oxygen-carrying vessels, re-examination has resulted in the discovery that RBCs do have interactions with the host immune system. Though mature RBCs lack the DNA and RNA for encoding and transcribing new proteins and the organelles required for signal transduction, their sheer number and routine transit between distant locations in the body make them uniquely qualified to convey signals of infection or danger between different organs and components of the immune system. From the first reports of immune adherence in 1917 to the discovery of Duffy Antigen Receptor for Chemokines (DARC) to our recent work demonstrating immune modulation via Toll-Like Receptor 9 (TLR9), it is becoming increasingly clear that RBCs are a critical component to host immune response[2–4]. The COVID-19 pandemic has highlighted the remarkable heterogeneity of the host response to a particular pathogen and the importance of the vascular compartment in the host response[5–7]. Thus, a better understanding of the innate immune response in the vascular compartment will be critical to furthering our knowledge of the heterogeneous host response. The last several years have witnessed unprecedented investigations into the non-gas exchanging role of RBCs, including nucleic acid binding, complement regulation, metabolism, and vascular regulation. This review will explore these significant advances in understanding RBC immune function to gain novel insights into the host response to pathogens and sterile inflammation.

Novel insights into interactions between RBCs and other host immune processes

Though it has long been demonstrated that the inflammatory response to infection impacts RBCs, the question remains as to what role RBCs actively play in initiating the immune response. Several recent papers show novel insights into the mechanism by which red blood cells not only interact with the host immune system but function as sentinels to trigger the inflammatory process.

One of the most critical components of the innate immune system is the ability to alert the body to the presence of toxic or non-self-material. This recognition occurs through evolutionarily conserved toll-like receptors (TLRs), located on the cell surface and intracellularly on diverse host cells that promote inflammatory cytokine secretion and immune cell responses[8–10]. Because nucleic acids are derived from host and pathogen, the nucleic acid sensing tolls are highly regulated and are generally located intracellularly. Our prior work demonstrated that RBCs express TLR9, allowing them to scavenge cell-free (CpG-containing) mitochondrial DNA (cf-mtDNA) under quiescent conditions.[11] However, the role of RBC-TLR9 during inflammatory states such as sepsis and the dysregulated response to infection remained unknown. We recently demonstrated the presence of TLR9 on the surface of RBCs and observed that the RBCs from critically ill patients with sepsis displayed increased surface TLR9 compared with healthy donors.[3]

Exposure of RBCs to high doses of CpG DNA resulted in morphologic changes, a reduction in osmotic fragility, and masking the antiphagocytic epitope of the “marker of self” CD47. CpG-binding by RBCs resulted in accelerated RBC clearance that RBC-TLR9 mediated. In a murine model of CpG-induced inflammation and polymicrobial sepsis, the loss of RBC-TLR9 resulted in attenuated IL-6 production in the tissues and plasma. These findings suggest that RBC-TLR9 drives red cell clearance and inflammatory cytokine production during sepsis through the delivery of CpG-DNA. Consistent with these experimental findings, patients with sepsis and COVID-19-associated sepsis demonstrated higher levels of RBC-associated mtDNA than healthy controls, which was associated with anemia.[3]

This data suggests that as cell-free CpG-DNA increases, such as in states of infection or sterile inflammation, it binds to RBCs in a TLR9-dependent manner, resulting in increased red cell clearance and concomitant innate immune activation. This work represents one of the first examples by which RBCs can trigger the host’s inflammatory response.

Another recently demonstrated mechanism for RBC immune function is the induction of vascular dysfunction. Mahdi et al. al. Incubated RBCs from hospitalized COVID-19 patients and healthy patients with rat aortic segments. They found that RBCs from COVID-19 patients (C19-RBCs) induced severe impairment in endothelial-dependent and endothelial-independent relaxation compared to healthy RBCs (h-RBCs)[12]. In investigating a method for RBC-induced vascular dysfunction, they found that C19-RBCs demonstrated increased ROS production and upregulated vascular arginase 1 in endothelial and smooth muscle cells. Direct inhibition of arginase 1 and superoxide attenuated the RBC-induced microvascular dysfunction. To better understand the RBC alterations present during COVID-19, healthy RBCs were incubated with IFNγ to mimic SARS-CoV2-induced inflammation. IFNγ treated h-RBCs induced impaired endothelial function similar to C-19 RBCs; the authors conclude that RBCs may be carriers of IFNγ during acute inflammatory states that may drive vascular dysfunction. These findings merit further investigation because IFNγ is a well-known inducer of endothelial cytotoxicity.

Insight into disease response: COVID-19

Proteomics, RBC structure

As the previous studies suggest, investigating RBC alterations in response to specific pathogens or cytokines may be necessary to better understand how RBCs drive downstream host responses, including vascular dysfunction. D’Alessandro et al. applied metabolomics, proteomics, and lipidomics approaches to compare RBCs banked from healthy patients to those of patients with COVID-19; the group found significant alterations to the RBC membrane[13]. The researchers demonstrated that RBCs from COVID-19 patients had elevated glycolytic intermediates, increased oxidative damage to structural membrane proteins, and altered lipid metabolism. The authors concluded that SARS-CoV2 infection altered RBC structure at the protein and lipid levels, seemingly “aging” the RBCs. The authors speculate that these alterations in RBCs of COVID-19 patients may contribute to their ability (or lack thereof) to cope with oxidant stress and hypoxemia.

Further evidence of physical changes to RBCs in response to COVID-19 came from Kubankova et al. They used real-time deformability cytometry (RT-DC) to investigate material changes in blood from healthy donors, patients with COVID-19 infection, and discharged patients who recovered from COVID-19. They found that RBCs from COVID-19 patients were smaller, though with increased variation in size, and had lower deformation [14]. They found that many of these structural changes persisted in recovered COVID-19 patients, perhaps providing insight into the underlying pathology of the “long COVID” phenomenon.

Similarly, Grua et al. compared RBC morphological parameters of patients after the acute phase of mild COVID-19 infection to healthy controls. They found that COVID-19-exposed RBCs had higher percentages of permanently elongated RBCs and membrane extension, altered MCH and MCV, and decreased RBC deformability in response to an osmotic gradient [15].

These studies of RBC alterations following SARS-CoV2 infection suggest that pathogen infection and consequent inflammation, even without direct RBC infection, can alter RBCS through secondary mediators. Additional exploration of RBC alterations during inflammatory states is warranted to determine if these findings are generalizable to other infectious and inflammatory conditions.

Complement Regulation

In addition to investigations into changes in RBC membrane structure, several recent works have investigated the interaction between COVID-19, RBCs, and the complement pathway. The complement pathway is one of the oldest components of the innate immune system. Because Complement Receptor type 1 (CR1) recognizes complement activation products C3b and iC3b and is more abundantly expressed on RBCs than anywhere else in the body. It has been investigated in several studies as a potential link between RBCs and pathogen response.[7,16]

A recent investigation by Kisserli et al. found that the density of CR1 on RBCs is decreased in patients with COVID-19 and that this decrease was related to the clinical severity of the disease.[7] This loss of CR1 density would predictably lead to a loss of anti-inflammatory complement inhibition, resulting in widespread complement activation and inflammation consistent with the disease phenotype of COVID-19. The group hypothesized that the mechanism behind this decreased density is a saturation of the CR1 receptors by complement fragment coated-virus and virus-containing immune complexes leading to the shedding of CR1 by the RBCs. This results in complement fragment accumulation on the RBCs, augmenting the immune response. While further investigation is needed into the exact mechanism of RBC involvement in the complement-driven immune response to SARS-CoV2, this study provides a clear link between RBCs and this ancient arm of the immune system.

A recent publication from our lab supported this study, finding that the RBCs of patients with both COVID and non-COVID Sepsis have increased C3b and C4d depositions compared to healthy controls.[16] This likely reflects the same process illustrated in the earlier study and provides another benefit to better understanding RBCs’ role in the immune response, improved diagnostics. While plasma-based assays of individual complement components have failed to reflect the levels present at the time of tissue injury, RBC flow cytometry may be a sensitive marker for complement activation. It could furthermore serve as a biomarker for severe COVID-19 disease progression and trajectory. Whether RBC bound complement fragments can be used to diagnose dysregulated complement activation in other inflammatory conditions, including autoimmune syndromes remains to be seen.

Conclusions

These studies over the past two and a half years contribute to the growing literature supporting the notion that RBCs have functions that far exceed their oxygen-transporting capabilities. They indicate a diverse array of mechanisms by which RBCs may both respond to a pathogen or sterile injury and drive the host immune response making further investigation into these processes essential to a comprehensive understanding of the innate immune response.

The need to better understand RBCs’ role in the host immune response is driven by the hypothesis that there is heterogeneity in how different hosts respond to the same pathogen. Consequently, a better understanding of the physiologic drivers for host heterogeneity will provide critical insights for developing precision medicine approaches to infectious diseases. Several recent studies of critically ill patients have demonstrated distinct phenotypes with different responses to protocolized therapies, highlighting the need for targeted therapies in heterogeneous inflammatory syndromes.[6,17–20] The collection and biobanking of RBC samples is a critical step in furthering the translational studies needed to better understand the immunomodulatory functions of RBCs. Given the rapidly accumulating evidence for RBC immune function and the recognition that the host immune response to pathogens is heterogeneous, research efforts into large-scale exploration of RBC immune function must be prioritized.

Key Points:

• RBCs modulate the host immune response through diverse mechanisms.

• Future studies of RBC immune function may provide insight into divergent host responses during inflammatory states.