Blandine Maître

In mammals, most of the iron is found in the heme of red blood cells (RBCs), which must be recycled to support erythropoiesis in the bone marrow. Splenic red pulp macrophages (RPMs) play a crucial role in this process by phagocytosing senescent RBCs, metabolizing the heme and releasing iron back into the blood. Free cytoplasmic iron generates toxic reactive oxygen species, yet iron-specific adaptations of RPMs are not well documented. We previously reported that autophagy prevents ferroptosis in Langerhans cells, a cutaneous phagocyte subset. Thus, we hypothesized that autophagy may be important for the regulation of RPM metabolism and their maintenance of systemic iron homeostasis. To study this, we used Atg5 ^flox/flox and Cd169 ^cre mouse models to delete ATG5 in CD169 ⁺ macrophages, including RPMs. Atg5 -deficient RPMs were decreased in number, and the remaining ones showed increased generation of toxic lipid peroxides. Spleens of Atg5 ^{Δ Cd169} mice were enlarged and contained more RBCs. Finally, autophagy impairment in RPMs exacerbated RBC loss in a model of phenylhydrazine-induced anemia. Our findings exemplify how dysregulation of macrophage metabolism alters their function and can disrupt tissue homeostasis upon challenge.

HLA class I–immunized patients can experience a serious complication known as platelet transfusion refractoriness (PTR). This issue becomes especially relevant in onco-hematology departments where platelet transfusions are at the heart of patient care. Although transfusion failure is evidenced by a rapid elimination of allogeneic platelets from the recipient’s bloodstream, the mechanisms behind it remain poorly characterized. The aim of this study was to better define these mechanisms to improve therapy for PTR. Using a murine model of major histocompatibility complex class I incompatibility to mimic PTR, we first established that antibodies, but not natural killer or CD8 cells, mediated platelet clearance. However, blocking Fcγ receptors with intravenous immunoglobulin or a monoclonal antibody or complement depletion did not correct refractoriness in alloimmune mice. Therefore, we investigated other alternatives beyond antibody-dependent mechanisms. Flow cytometric and microscopic analysis showed that Kupffer cells in the liver and red pulp macrophages in the spleen phagocytose allogeneic platelets during PTR. Moreover, intravital microscopy revealed allogeneic platelets retained in close interaction with macrophages in the red pulp only in alloimmune animals. Splenectomy or Kupffer cell depletion with clodronate in alloimmune mice suggested the existence of compensatory elimination mechanisms in the liver and spleen. Therefore, the simultaneous removal of both macrophage populations was an effective strategy to abrogate PTR. Our study provides an insight into the mechanisms of platelet clearance in alloimmune pathologies and opens up new perspectives for therapeutic targets.

Patients requiring recurrent platelet transfusions are subject to platelet transfusion refractoriness (PTR), a therapeutic failure due to rapid clearance of transfused platelets from the circulation. One major cause of PTR is the presence in the recipient of multiple IgG directed against allogeneic HLA Class-I (HLA-I) molecules expressed by the donor platelets. Strikingly, the presence of anti-HLA-I IgG does not necessarily correlate with PTR, thus questioning the role of the antibody properties themselves. Using blood of HLA-I alloimmunized patients with or without PTR, we identified the subclasses of their anti-HLA-I IgG. We found the distribution of these subclasses to be different according to patients, with IgG1 being predominant in non-PTR patients while IgG1 in combination with IgG2 or IgG3 were detected in PTR patients. To understand the mechanisms associated with PTR, we used human chimeric pan-HLA-I IgG1, IgG2, or IgG3 antibodies and assessed the functional implications of these human IgG subclasses on platelet activation. We showed that each subclass led to platelet aggregation, P-selectin exposure and Annexin V binding. However, we found that the mechanisms of platelet activation differed between subclasses. Specifically, we discovered that pan-HLA-I hIgG2-induced platelet activation was CD32a dependent, while hIgG1- and hIgG3-induced platelet activation relied on complement recruitment. Hence, this study may have direct implications for hierarchizing pathogenic anti-HLA-I alloantibodies in highly polyimmunized patients and be a valuable aid in selecting suitable treatments, particularly by streamlining the search for functionally compatible platelet components.

Alloimmunization against platelets remains a potentially serious adverse transfusion event. Alloantibodies produced by the recipient, mainly directed against human leukocyte antigen class I donor antigens, can compromise the therapeutic efficacy of subsequent transfusions, and may lead to refractoriness. Because the mechanism of anti-HLA alloantibody formation is poorly understood, this study aimed to identify the cells involved in the platelet immune response by focusing on the spleen, the main organ that orchestrates this alloimmune response. In the spleen, transfused allogeneic platelets are located in the marginal zone and interact with marginal zone B (MZB) cells, a specialized B-cell population implicated in the capture and follicular delivery of blood-borne antigens. To study the involvement of MZB cells in alloantibody production, we used a murine model reproducing major histocompatibility complex incompatibility between a donor (H2b) and recipient (H2d) that occurs during platelet transfusion. Following weekly H2b platelet transfusions, recipient H2d mice produced anti-H2b immunoglobulin G, which induced a refractory state upon subsequent transfusions. Specific immunodepletion of MZB cells or their displacement from the marginal zone to the B-cell follicles by treatment with an S1P1 antagonist before each transfusion prevented significant alloantibody formation. Under these conditions, transfused platelets were still circulating after 24 hours, whereas they were rapidly removed from circulation in alloimmunized mice. The identification of MZB cells as key players in the platelet alloimmune response opens up new perspectives for minimizing platelet alloimmunization and avoiding the associated refractory state in frequently transfused patients.

Patients with hematological disorders and severe thrombocytopenia require extensive and iterative platelet transfusion support. In these patients, platelet transfusion refractoriness represents a serious adverse transfusion event with major outcomes for patient care. Recipient alloantibodies against the donor HLA Class I antigens expressed at the cell surface of platelets result in a rapid removal of transfused platelets from the circulation and thus, therapeutic and prophylactic transfusion failure leading to a major bleeding risk. In this case, the only way to support the patient relies on the selection of HLA Class I compatible platelets, an approach restricted by the limited number of HLA-typed donors available and the difficulty of meeting the demand in an emergency. However, not all patients with anti-HLA Class I antibodies develop refractoriness to platelet transfusions, raising the question of the intrinsic characteristics of the antibodies and the immune-mediated mechanisms of platelet clearance associated with a refractory state. In this review, we examine the current challenges in platelet transfusion refractoriness and detail the key features of the antibodies involved that should be considered. Finally, we also provide an overview of future therapeutic strategies.

Mice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2β1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2β1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2β1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2β1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.

The biological responses that control the development of Transfusion-Related Acute Lung Injury (TRALI), a serious post-transfusion respiratory syndrome, still need to be clarified. Since extracellular nucleotides and their P2 receptors participate in inflammatory processes as well as in cellular responses to stress, we investigated the role of the ATP-gated P2X₁cation channel in antibody-mediated TRALI. The effects of NF449, a selective P2X1 receptor (P2RX1) antagonist, were analyzed in a mouse two-hit model of TRALI. Mice were primed with lipopolysaccharide (LPS) and 24 h later challenged by administrating an anti-MHC I antibody. The selective P2RX1 antagonist NF449 was administrated before the administration of LPS and/or the anti-MHC I antibody. When given before antibody administration, NF449 improved survival while maximal protection was achieved when NF449 was also administrated before the sensitization step. Under this later condition, protein contents in bronchoalveolar lavages were dramatically reduced. Cell depletion experiments indicated that monocytes/macrophages, but not neutrophils, contribute to this effect. In addition, the reduced lung periarteriolar interstitial edemas in NF449-treated mice suggested that P2RX1 from arteriolar smooth muscle cells could represent a target of NF449. Accordingly, inhibition of TRPC6, another cation channel expressed by smooth muscle cells, also reduced TRALI-associated pulmonary interstitial and alveolar edemas. These data strongly suggest that cation channels like P2RX1 or TRPC6 participate to TRALI pathological responses.