C5a Blockade Increases Regulatory T Cell Numbers and Protects Against Microvascular Loss and Epithelial Damage in Mouse Airway Allografts

Abstract

Microvascular injury during acute rejection has been associated with massive infiltration of CD4+ T effector cells, and the formation of complement products (C3a and C5a). Regulatory T cells (Tregs) are potent immunosuppressors of the adaptive immune system and have proven sufficient to rescue microvascular impairments. Targeting C5a has been linked with improved microvascular recovery, but its effects on the Treg and T effector balance is less well known. Here, we demonstrate the impact of C5a blockade on Treg induction and microvascular restoration in rejecting mouse airway allografts. BALB/c→C57BL/6 allografts were treated with a C5a-neutralizing l-aptamer (10 mg/kg, i.p. at d0 and every second day thereafter), and allografts were serially monitored for Treg infiltration, tissue oxygenation (tpO2), microvascular blood flow, and functional microvasculature between donor and recipients during allograft rejection. We demonstrated that C5a blocking significantly leads to enhanced presence of Tregs in the allograft, reinstates donor–recipient functional microvasculature, improves tpO2, microvascular blood flow, and epithelial repair, followed by an upregulation of IL-5, TGF-β, IL-10 vascular endothelial growth factor, and ANGPT1 gene expression, while it maintained a healthy epithelium and prevented subepithelial collagen deposition at d28 posttransplantation. Together, these data indicate that inhibition of C5a signaling has potential to preserve microvasculature and rescue allograft from a sustained hypoxic/ischemic phase, limits airway tissue remodeling through the induction of Treg-mediated immune tolerance. These findings may be useful in designing anti-C5a therapy in combination with existing immunosuppressive regimens to rescue tissue/organ rejection.

Complement factor and T-cell interactions during alloimmune inflammation in transplantation

“…Further, studies with adoptive transfer of T cells from WT mice into C3aR −/− and C5aR −/− mice supported the notion that ablation of C3aR and C5aR mediated signaling to affect the complement and T‐cell interaction and subsequent downregulation of costimulatory molecules and IL‐6, while upregulation of IL‐10 and systemic Tregs generation during alloimmune inflammation . Recently, targeted blockade of complement C5a was found sufficient to induce peripheral and graft CD4 + FOXP3 + Tregs and augmented the microvascular blood flow between donor and recipients grafts, which resulted in suppression of subepithelial collagen deposition during rejection . These findings supported the notion that C5a blockade, anti‐C5a‐C5aR ligation, shifted the immune balance toward Treg phenotype, which favored the reparative phase through the secretion of various cytokines (IL‐10, IL‐5, and TGF‐β) .…”

“…A number of preclinical and clinical studies have identified crucial roles of active complement proteins, such as C1q, C3b/iC3b, C3a (and C3aR), and C5a (and C5aR) in T‐cell response during homeostasis and disease, which affect T‐ cell function via APCs, the cytokine milieu, or downstream complement activation products . The release of C3, fB, and fD, as well as C5, and upregulation of C3aR and C5aR have been reported during cognate interactions between T cells and APCs (macrophages and DCs), which are induced through CD28/CD80/CD86 and CD154/CD40 molecular signaling on the membrane of APCs . Considering the involvement of complement‐mediated costimulation of T cells and development of new complement inhibitors suggested that the therapeutic use of costimulation blockade through complement inhibitors has been under preclinical and clinical testing to improve graft injuries while minimizing global suppression of protective immunity, which could become a valuable therapeutic step for transplant patients in combination therapies.…”

“…Various studies highlighting complement interactions with the various inflammatory cells through corresponding receptors allow an insight into the molecular details of complement‐mediated transplant injuries and terminal graft failure during rejection . Despite significant therapeutic advancements, the permanent cure of graft rejection is still not achieved, which involve a series of molecular and cellular interactions that lead to irreversible chronic transplant failure .…”

“…Tregs are crucial in maintaining immunotolerance and have been associated with improved long‐term transplant survival, as seen in various preclinical and clinical transplant studies . Complement and T‐cell interactions have been key to influence alloimmune state, which initiated through the activation of complement cascade, and their subsequent interactions with neighboring inflammatory cells have been critical to transplant‐associated injuries, which result in obliteration of graft microvasculature, and ultimately leads to the progression of airway fibrosis and chronic rejection . On the flip side, Tregs also display surface expression of various complement receptors, and binding of these receptors with corresponding activated complement fragments significantly affects the outcomes of the regulatory microenvironment of transplant.…”

“…During an effective alloimmune reaction, activated immune cells mount a massive inflammatory environment via inflammatory mediators (cytokines, leukotrienes), and through interactions with various cleaved products (C3a and C5a) of complement cascade . T cell has been recognized as one of the major cells to initiate an exuberant alloimmune response to foreign antigens during graft rejection, but its interaction with activated complement factors further bolster the effectiveness of an alloimmune response during rejection phase, which involves microvascular associated injuries, tissue remodeling, and progression of fibrosis . It has been well‐demonstrated that activated complement factors, CD4 + T cells and CD8 + T cells, are expressed on a vascular endothelium and contribute to microvascular injury to reject grafts through the endothelial‐derived complement activation .…”