Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration

Nathalie Cloutier; Isabelle Allaeys; Genevieve Marcoux; Kellie R. Machlus; Benoit Mailhot; Anne Zufferey; Tania Levesque; Yann Becker; Nicolas Tessandier; Imene Melki; Huiying Zhi; Guy Poirier; Matthew T. Rondina; Joseph E. Italiano; Louis Flamand; Steven E. McKenzie; Francine Cote; Bernhard Nieswandt; Waliul I. Khan; Matthew J. Flick; Peter J. Newman; Steve Lacroix; Paul R. Fortin; Eric Boilard

doi:10.1073/pnas.1720553115

New Research In

Edited by Barry S. Coller, The Rockefeller University, New York, NY, and approved January 5, 2018 (received for review December 1, 2017)

Significance

Immune complexes (ICs) form when antibodies encounter their antigens. ICs are present in blood in multiple pathological conditions. Given the abundance of platelets in blood and that they express a receptor for ICs, called Fcγ receptor IIA (FcγRIIA), we examined the impact of ICs in blood in a mouse model. We found that circulating ICs induced systemic shock, characterized by loss of consciousness, by activating platelet FcγRIIA. Shock was mediated by the liberation of serotonin, a molecule better known for its role in the brain, from platelet granules. During shock, platelets were sequestered in the lungs and brain and returned to the blood circulation after their degranulation. Platelets are thus crucial in response to ICs.

Abstract

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood–brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Platelets are best known for their involvement in hemostasis. Their abundance in blood underlies their perfect positioning for constant surveillance of the endothelium to ensure vascular integrity. The role of platelets, however, is not restricted to the hemostatic response (1). Not only do platelets express a vast array of mediators serving wound repair but they also possess immune receptors and inflammatory molecules (1 ⇓–3). Moreover, following tissue injury or infection, platelets are a central effector in driving an inflammatory process for recruiting leukocytes, principally neutrophils, to the affected site (4 ⇓–6). The platelet contribution at an inflamed site also includes the prevention of bleeding (7, 8), as platelets seal the breaches formed during the extravasation of neutrophils (9).

Platelet activation occurs at the interface with the vasculature, is elicited by injury or inflammation, and is restricted to the endothelium or tissue. However, given the great number of platelets in blood and their extensive set of immune receptors, systemic immune triggers can also activate platelets while in circulation (i.e., in the absence of endothelial or tissue injury). In rheumatic diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and antiphospholipid syndrome, there is a prevalence of pathogenic antibody–antigen scaffolds, called immune complexes (ICs), in circulation in patients (10). Anaphylactic reactions due to allergens (11, 12) and i.v. administration of drugs, such as in heparin-induced thrombocytopenia (HIT) (13), also implicate ICs, thereby provoking acute inflammatory responses and a rapid drop in platelet count (thrombocytopenia). ICs can also form in blood during sepsis and viremia, when antibodies in the immune host recognize microbial antigens or their toxins.

The pathological link between ICs and cellular responses is mediated by members of the Fcγ receptor (FcγR) family. The low-affinity receptor FcγRIIA is the sole FcγR expressed by platelets in humans, and is thereby the most abundantly expressed receptor in blood (12, 14, 15). Of note, it is the dominant receptor activated by Zika virus if in the presence of sera from convalescent patients infected by dengue or West Nile virus, pointing to the formation of ICs in blood in patients with preexisting antiflavivirus immunity (16). Moreover, diverse strains of bacteria and the influenza virus H₁N₁ trigger platelet FcγRIIA activation after forming ICs in an immune host (17, 18). In addition, studies with human platelets show that ICs from patients with SLE also activate platelets through FcγRIIA (19). However, mice do not express FcγRIIA, and murine platelets are completely devoid of any FcγRs (12). Thus, IC-mediated responses in mice strongly favor leukocytes, which express other FcγRs, while platelets play small or accessory roles in mouse models implicating ICs.

To more accurately model FcγRIIA reactions in mice, transgenic FcγRIIA (FcγRIIA^TGN) mice that display FcγRIIA expression on platelets and certain leukocytes, including monocytes and neutrophils as in humans, were developed (12, 20). Notably, stimulation of FcγRIIA in transgenic mice induces a shock response that is reminiscent of anaphylaxis in humans, with a strong reliance on neutrophil activity and implicating release of platelet-activating factor (PAF) (14, 21 ⇓⇓–24).

How ICs trigger systemic responses is not completely understood, as no study has definitively determined the contribution of platelets to this response in vivo. Here, we employed FcγRIIA^TGN mice to elucidate the precise mechanisms by which platelets contribute to IC-mediated shock.

Results

Platelets Are Critical During the Systemic Response.

ICs injected into FcγRIIA^TGN mice trigger systemic shock (22 ⇓–24). Although thrombosis in the lungs and thrombocytopenia accompany systemic shock in these mice (22, 25 ⇓–27), the platelet contribution to IC-induced shock has never been formally assessed. We used heat-aggregated (HA) IgG as an IC surrogate (28, 29), as it permits precise investigation of the role of FcγR activation with no bias owing to the nature of antigen specificities. HA-IgG (160 ± 7 nm in diameter) was injected i.v. into WT mice lacking FcγRIIA (FcγRIIA^null) and FcγRIIA^TGN mice. Significant temperature loss (15 min) preceded by rapid significant systemic shock (3 min) in all of the FcγRIIA^TGN mice was confirmed, but it was never observed in the FcγRIIA^null mice (Fig. 1A and Movie S1). All of the FcγRIIA^TGN mice collapsed, underwent profound immobility, and lost consciousness (Fig. S1A). Mice recovered from the shock and hypothermia within 180 min, after which no residual signs were apparent (Fig. S1B). The occurrence of shock was dependent on the concentration of ICs present in blood, not induced by monomeric IgG, similarly induced when murine ICs were used, and more profound in male FcγRIIA^TGN mice compared with females (Fig. S1 C–E).

Fig. 1.

Platelets are critical during the systemic response. Systemic shock (Upper) (scores defined in Materials and Methods) and temperature (Lower) were measured at the indicated time points following IC injection in FcγRIIA^TGN and FcγRIIA^null mice (A; n = 12); FcγRIIA^TGN mice preinjected with platelet-depleting antibody (PLT Ab) and isotypic control antibody (Ctrl Ab) (B; n = 8); FcγRIIA^TGN/β3^+/+ and FcγRIIA^TGN/β3^−/− mice (C; n = 15); bone marrow chimeric mice generated by transfer of FcγRIIA^TGN cells into WT (D and E; native fibrinogen), Fibγ^∆5 (D), and Fibγ^390-396A (E) irradiated mice (n = 6); FcγRIIA^TGN mice preinjected with GPIb Fab (Xia.B2) or diluent (F; n = 6); FcγRIIA^TGN mice pretreated with aurintricarboxylic acid (ATA) or diluent (G; n = 8); and FcγRIIA^TGN mice pretreated with alteplase (H; n = 4) or diluent. null, FcyRIIAnull; TGN, FcyRIIATGN. Data are mean ± SEM. **P < 0.005, ***P < 0.001, and ****P < 0.0001; repeated-measures two-way ANOVA, statistical variation between groups (A–H).

To formally assess the contribution of platelets to the systemic shock, we depleted platelets (>98%) before injection of ICs. Thrombocytopenic mice were completely protected from IC-mediated systemic responses (Fig. 1B and Fig. S1F), identifying a critical role for platelets in shock induced by circulating ICs. In platelets, efficient αIIbβ3 signaling relies on the FcγRIIA immunoreceptor tyrosine-based activation motif, and, accordingly, the expression of FcγRIIA by platelets enhances fibrinogen-mediated platelet activation (30). Thus, we verified the functional association of FcγRIIA and the αIIbβ3 receptor in vivo by comparing the response in FcγRIIA^TGN/β3^−/− and FcγRIIA^TGN/β3^+/+ mice. The FcγRIIA^TGN/β3^−/− mice were completely resistant to IC challenge (Fig. 1C), further emphasizing the role of platelets and pointing to the requirement of αIIbβ3 in the mechanism of IC-mediated shock.

While αIIbβ3 may amplify FcγRIIA signaling through common kinases located in the platelet cytoplasm, it is predominantly known as the fibrinogen receptor, which may contribute upon binding to platelet activation (31 ⇓–33). Fibrinogen also mediates platelet–neutrophil interactions by bridging αIIbβ3 and macrophage-1 antigen (Mac-1) (34). Hence, platelet–neutrophil aggregates formed in blood in the presence of ICs (Fig. S2A), as well as neutrophils, were also necessary in the systemic responses (Fig. S2B), in agreement with prior studies (23, 28). To verify the specific role of fibrinogen, we used mutant fibrinogen chimera mice. We lethally irradiated fibrinogenγ^Δ5 (Fibγ^Δ5) and fibrinogenγ^390-396A (Fibγ^390-396A) mutant mice, which express mutant fibrinogen unable to bind αIIbβ3 or Mac-1 (34, 35), respectively, and engrafted the mice with bone marrow from FcγRIIA^TGN mice. After injection with ICs, we found that fibrinogen binding to αIIbβ3 was necessary for the systemic response, whereas bridging platelets with leukocytes through fibrinogen binding to Mac-1 was dispensable (Fig. 1 D and E). Consistent with these data, the blockade of Mac-1 as well as PSGL-1, the counterreceptor of platelet P-selectin on leukocytes (36), had no effect on shock (Fig. S2C).

Like αIIbβ3, GPIb is critical in the prevention of bleeding and thrombosis, and the ablation of the gene coding for GPIb in mice leads to severe bleeding defects (37). As GPIb is localized within lipid raft membrane microdomains in physical proximity with FcγRIIA (38), we used a GPIb-blocking Fab to probe the contribution of GPIb, and found that it is dispensable in shock mediated by ICs (Fig. 1F). Furthermore, the pharmacological blockade of von Willebrand factor (vWF) binding to GPIb (ATA; aurintricarboxylic acid) or administration of recombinant tissue plasminogen activator (tPA; alteplase) to promote the lysis of any existing thrombi also had no effect on shock (Fig. 1 G and H). These data further demonstrate that platelet activation by circulatory ICs implicates pathways distinct from those promoting vaso-occlusion and vascular injury.

Identification of the Shock Mediator.

Consistent with the rapid response, an increase in platelet factor 4 (PF4) and serotonin, components stored in α- and δ-granules, respectively, was detected in blood within 10 min (at the time of mouse collapse and before hypothermia) of the IC trigger in FcγRIIA^TGN mice (Fig. 2A). We therefore hypothesized that a component released from platelet granules may be responsible for induction of systemic shock. Intravital microscopy of the microvasculature using two-photon microscopy revealed significant vessel leakage and vasodilatation in response to IC injection in FcγRIIA^TGN mice (Fig. 2 B and C). Vessel leakage was systematically observed in response to IC injection in both FcγRIIA^TGN mice and FcγRIIA^null mice, but was absent in FcRγ^−/− mice, pointing to the role of other receptor(s) for ICs (Fig. S3A). It was also maintained in the absence of neutrophils, further suggesting that vascular leakage is not the key pathological feature downstream of FcγRIIA driving IC/platelet-mediated shock (Fig. S3A).

Fig. 2.

Serotonin is the mediator of shock. (A) PF4 (Left) and serotonin (Right) concentrations were measured in platelet-free plasma prepared from blood of FcγRIIA^TGN mice collected 10 min after IC injection (n = 5). The baseline (measured in nonchallenged FcγRIIA^TGN mice) concentration is indicated using a dotted line. Dil, diluent. (B and C) Intravital microscopy reveals profound changes to the mouse ear vasculature in response to ICs. (B) Vascular leakage, evidenced by the presence of Evans Blue (magenta) outside blood vessels in the subendothelial matrix rich in collagen (blue), is represented using FcγRIIA^TGN. (Scale bar: 75 μm.) (C) Vasodilatation was measured prior to leakage (t = 8 min) in FcγRIIA^TGN, FcγRIIA^null, or FcγRIIA^TGN/Tph1^−/− mice (n > 5 vessels per field in three mice per group). (D–G) Systemic shock (Upper) and temperature (Lower) were measured at the indicated time points following injection of serotonin (Sero) or its Dil in FcγRIIA^null mice (D; n = 6), after IC injection in FcγRIIA^TGN mice treated or not treated with the SSRI fluoxetine (E; n = 10), after IC injection in FcγRIIA^TGN/Tph1^+/+ and FcγRIIA^TGN/Tph1^−/− mice (F; n = 9), and after injection of ICs in FcγRIIA^TGN mice pretreated with the 5-hydroxytryptamine receptor 2 blocker ketanserin or Dil (G; n = 5). null, FcyRIIAnull; TGN, FcyRIIATGN. Data are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001, using an unpaired t test (A); one-way ANOVA (C); and repeated-measures two-way ANOVA, statistical variation between groups (D–G).

In stark contrast, vasodilatation was a response observed exclusively in FcγRIIA^TGN mice (Fig. 2C). Identified first as a serum agent mediating vascular tone, serotonin is a powerful vasoconstrictor when added to smooth muscle cells. However, it is a potent vasodilator on endothelial cells (39). In the case of systemic activation in the absence of vascular injury, and thus an intact endothelium, we hypothesized that serotonin might be the key platelet component driving vasodilatation. This is supported by the fact that the majority of peripheral serotonin, which represents 95% of the total body serotonin pool, is stored in platelet δ-granules (40, 41). Platelets do not synthesize serotonin; they utilize their serotonin transporter (SERT) to capture circulatory serotonin, with the latter being generated by enterochromaffin cells from the digestive tract by the enzyme tryptophan hydroxylase 1 (Tph1) (42, 43). Consistent with our hypothesis, vasodilatation was abrogated in FcγRIIA^TGN/Tph1^−/− mice in response to ICs (Fig. 2C). It was also maintained in absence of neutrophils, further confirming the direct role of peripheral serotonin in IC-induced vasodilatation of microvessels (Fig. S3B).

Serotonin levels in plasma were back to normal 1 h postshock (Fig. S3C). To directly verify its role in systemic shock, serotonin was injected into FcγRIIA^null mice, which resulted in a shock response reminiscent of the responses observed in IC-injected mice (Fig. 2D). We then used a selective serotonin reuptake inhibitor (SSRI), a SERT blocker used as an antidepressant, to inhibit serotonin storage by platelets. Administration of the SSRI for 3 wk to deplete the serotonin content of δ-granules (41) revealed the important contribution of serotonin uptake in IC-mediated inflammation: SSRI treatment nearly abolished the systemic shock response (Fig. 2E). Furthermore, FcγRIIA^TGN/Tph1^−/− mice lacking platelet serotonin (43) showed almost complete resistance to shock (Fig. 2F), confirming the critical role of peripheral serotonin in IC-mediated shock. In addition, blockade of the 5-hydroxytryptamine 2 receptor family, which is expressed in the periphery by platelets, but also by monocytes and macrophages, dendritic cells, eosinophils, B and T lymphocytes, endothelial cells, fibroblasts, cells from the cardiovascular system, and neurons in the peripheral nervous system (40), was effective at reducing shock (Fig. 2G). In support of the potential role of αIIbβ3 in degranulation, serotonin was not released in FcγRIIA^TGN/β3^−/− mice after IC injection, in agreement with the absence of shock in those mice (Fig. S3D). Key actors in the prevention of bleeding and thrombosis, the platelet-derived mediators ADP and thromboxane A₂, were dispensable for shock (Fig. S3 E and F). In addition, neutrophil extracellular traps, which can be present in thrombosis (44), were detected during shock according to quantifications of circulating nucleosomes (Fig. S3G); however, the injection of DNase did not protect mice from shock (Fig. S3H). Together, these observations highlight the importance of platelet-derived serotonin in the systemic response to ICs in vivo, a platelet response that is distinct from that traditionally observed in hemostasis and thrombosis.

And the Platelet Count?

Circulating platelets were also monitored throughout the systemic response and beyond. We observed that FcγRIIA^TGN mice rapidly underwent profound thrombocytopenia (∼10% of total normal platelet count) in response to ICs, whereas FcγRIIA^null mice presented with only very minimal or no changes in platelet counts (Fig. 3A). In fact, thrombocytopenia occurred rapidly (<10 min), and the platelet count gradually increased, reaching ∼20% by 60 min, and it was 50% resolved within 24 h (Fig. 3A). The occurrence of thrombocytopenia was critically dependent on the expression of FcγRIIA and was similarly induced in males and females (Fig. S4A). Only platelets were affected, as the levels of RBCs, monocytes, and neutrophils were unchanged in response to ICs (Fig. S4 B–D). Thrombocytopenia was as profound in native fibrinogen mice as in Fibγ^Δ5 and Fibγ^390-396A mice (Fig. 3B), suggesting that fibrinogen binding was dispensable, and that thrombocytopenia only modestly involved β3, potentially through binding to its other ligands. Conversely, thrombocytopenia was unaltered by the inhibition of GPIb and vWF interactions, or the blockade of thromboxane A₂ synthesis and ADP (Fig. 3B). Thrombin–antithrombin complexes and D-dimers, which are evidence of coagulation activation and thrombus degradation, respectively, were not significantly elevated 24 h after shock (Fig. S4 E and F), and consistent with this, the destruction of potential thrombi by the injection of alteplase did not impact platelet count (Fig. 3B). These data further dissociate the platelet response in coagulation and thrombosis from IC-induced thrombocytopenia.

Fig. 3.

Thrombocytopenia in response to ICs. (A and B) Blood samples were collected at the indicated time points following IC injection, and CD41⁺ platelets in whole blood were counted using a flow cytometer. (A) Platelet count was determined in FcγRIIA^TGN and FcγRIIA^null mice (n = 11). (B) Percentages of circulating platelets 10 min after IC injection compared with initial platelet count were calculated in FcγRIIA^null mice (black) and the indicated three different groups of FcγRIIA^TGN mice. Mice were pretreated with acetylsalicylic acid (ASA) (n = 4), apyrase (n = 6), aurintricarboxylic acid (ATA) (n = 3), alteplase (n = 5), or diluent (n = 14) (all shown in blue). FcγRIIA^TGN mice pretreated with GPIb Fab or control (n = 4), FcγRIIA^TGN/β3^+/+ or FcγRIIA^TGN/β3^−/− mice (n = 10); FcγRIIA^TGN/Tph1^+/+ mice; or FcγRIIA^TGN/Tph1^−/− mice (n = 4) are represented (all shown in purple). Bone marrow chimeric mice generated by transfer of FcγRIIA^TGN cells into WT (native fibrinogen), Fibγ^∆5, and Fibγ^390-396A irradiated (IRRAD) mice are represented (n = 3 per group) (shown in green). (C and D) Mice were injected with diluent or IC, and blood was collected 24 h later. (C) Exposition of PS and P-selectin (P-sel) was assessed on CD41⁺ platelets using flow cytometry in FcγRIIA^TGN mice (n = 7). (D) Platelet content of PF4 (n = 6) and serotonin (n = 5) was measured by ELISA in 10⁶ platelets retrieved 24 h after shock. Baseline (measured in nonchallenged FcγRIIA^TGN mice) contents are indicated using dotted lines. (E) PF4 and serotonin-negative cells were counted using immunofluorescence microscopy (n = 3 different mice). Representative image of Z-stack projections using confocal microscopy. Platelets that returned to circulation 24 h after IC injection were used for quantification. Tubulin (red) was used as a platelet marker. PF4 (green) and serotonin (green) were observed in less than 60% of platelets. Empty platelets (arrowheads) and platelets (arrows) are represented. (Scale bars: 2 μm.) As a negative control, serotonin labeling was performed on Tph1^−/− platelets (Fig. S9). (F) Electron microscopy of FcγRIIA^TGN platelets before IC injection (Upper) and 24 h after IC injection (Lower). Empty platelets (arrowheads) and platelets (arrows) are represented. (Scale bars: 0.8 μm.) (G) Systemic shock was measured in FcγRIIA^TGN mice injected with ICs at t = 0 and rechallenged at t = 24 h (n = 3). (H) Plasma levels of PF4 (n = 4) and serotonin (n = 5) were determined in mice rechallenged 24 h after the first challenge with ICs. Results were compared with the level after the first challenge (dotted lines). (I) Platelets were purified from FcγRIIA^TGN mouse blood, fluorescently labeled, and adoptively transferred i.v. in FcγRIIA^TGN mice. One hour later, mice were injected with ICs, and fluorescent platelets in circulation were determined at the indicated time points following injection of ICs (n = 10). Dil, diluent; null, FcyRIIAnull; TGN; FcyRIIATGN. Data are mean ± SEM. **P < 0.005, ***P < 0.001, and ****P < 0.0001, using an unpaired t test (A, D, and H); one-way ANOVA (B, C, E, and I); and repeated-measures two-way ANOVA, statistical variation between groups (G).

Platelet conversion to microparticles also did not explain the profound thrombocytopenia, as microparticle levels only increased 60 min after the IC trigger (Fig. S4G). The decrease in platelet number could also not be attributed to platelet interaction with leukocytes, as neutrophil depletion, or blockade of Mac-1 and PSGL-1, had no effect on thrombocytopenia (Fig. S4 H and I). Given that FcγRIIA^TGN/Tph1^−/− mice also underwent profound thrombocytopenia (Fig. 3B), and that exogenous serotonin did not induce thrombocytopenia (Fig. S4J), this suggests that serotonin serves as the effector of IC-mediated shock but is not the driver of the resulting thrombocytopenia. Thus, although thrombocytopenia occurs concurrently with shock, it is not causative in the mechanism of inducing the shock.

The unanticipated rapid recovery in platelet count prompted a detailed analysis of the platelets present in blood 24 h after IC challenge. We found that platelets were not activated or apoptotic, as evidenced by the lack of phosphatidylserine (PS) and P-selectin on their surface (Fig. 3C). However, the platelet granule content was markedly reduced (Fig. 3D), as ∼30% of the circulating platelets contained no detectable serotonin or PF4 (Fig. 3E), suggesting significant degranulation. Electron microscopy further confirmed that circulating platelets were frequently devoid of any granules (Fig. 3F). Notably, platelets still expressed surface FcγRIIA 24 h postshock (Fig. S5 A and B), and still underwent thrombocytopenia if challenged a second time with ICs (Fig. S5C). This is in contrast to shock, where FcγRIIA^TGN mice were resistant to shock induced by further challenges (Fig. 3G), and neither serotonin nor PF4 was induced in the blood of these mice (Fig. 3H). Thus, we hypothesized that platelets circulating 24 h postshock were, in fact, platelets that had already degranulated and had undergone temporary sequestration. To verify this, we performed fluorescent labeling of FcγRIIA^TGN platelets, which we adoptively transferred into FcγRIIA^TGN mice. As expected, 87 ± 12% of fluorescent platelets, which were negative for PS (Fig. S5D), rapidly became undetectable from the blood circulation after an IC trigger. Of particular importance, 42 ± 16% of the fluorescently labeled FcγRIIA platelets, devoid of any surface PS, were identified in blood 24 h after shock (Fig. 3I), confirming a temporary sequestration of platelets following FcγRIIA activation and their return to the blood circulation after degranulation.

Localization of Sequestration Sites.

We next aimed to determine the sites of platelet sequestration in response to FcγRIIA activation. An intravital imaging system (IVIS) provided evidence of fluorescently labeled platelets in the lungs (Fig. 4A), but not in other locations, in agreement with previous studies (22, 25 ⇓–27). Platelet thrombi populated with neutrophils were also evident in the lungs (Fig. 4B), although no pulmonary edema was observed (Fig. 4C). Thrombi contribution to platelet sequestration and shock was not significant: ablation of the β3 gene, or blockade of GPIb using Fab, significantly reduced thrombus formation in FcγRIIA^TGN mice (Fig. 4D) with only a modest or no impact on thrombocytopenia, respectively (Fig. 3B). Furthermore, serotonin had no effect on thrombus formation, as the number of thrombi in the lungs remained unchanged in FcγRIIA^TGN/Tph1^−/− mice (Fig. 4D). These data identify the lungs as the apparent major site for platelet sequestration, and highlight that although thrombi are present in the lungs, these thrombi play no role in the thrombocytopenia and shock mediated by ICs.

Fig. 4.

Localization of sites of sequestration. (A) Mouse platelets from whole blood of FcγRIIA^TGN mice were labeled in vivo before IC injection. The kidneys (Kid), spleen (Spl), liver (Liv), heart (He), lungs (Lu), and brain (Br) were harvested 10 min after IC injection, and fluorescence was measured in each organ using an IVIS to determine platelet localization (n = 3). Results were compared with FcγRIIA^null mice injected with diluent, and are presented as the percentage of fluorescence in diluent-injected FcγRIIA^null mice. (B) Lungs were collected 10 min after IC injection. Thrombi (star) and neutrophils (arrows) were examined by microscopy after hematoxylin and eosin coloration. (Magnification: 400×.) (C) FcγRIIA^TGN mice were injected with diluent (Dil) or ICs, and lungs were collected after 10 min. The lung wet-to-dry ratio was evaluated. (D) Number of lung thrombi per square millimeter was quantified in FcγRIIA^null (n = 4), FcγRIIA^TGN (n = 12), FcγRIIA^TGN/β3^−/− (n = 7), and FcγRIIA^TGN/Tph1^−/− (n = 3) mice, as well as in FcγRIIA^TGN mice pretreated with GP1b Fab antibody (n = 4). (E) Mouse platelets from FcγRIIA^null and FcγRIIA^TGN mice were labeled in vivo. Lungs were collected 10 min after the IC trigger, and the number of fluorescent platelets was estimated (n = 4) using a standard curve designed with known numbers of fluorescent platelets spiked into nonfluorescent control lung homogenates. Percentages were obtained by comparison with platelet count obtained before the experiment and are presented as the percentage of total platelet number in the whole-mouse body. (F) ICs were labeled with DyLight-647 anti-Human IgG (fluo ICs) before injection in mice. The Kid, Spl, Liv, He, Lu, and Br were harvested 10 min later, and fluorescence was measured in each organ using an IVIS to determine IC localization (n = 3). Results are presented as the percentage of fluo determined in FcγRIIA^null mice. (G) Two-photon intravital microscopy in brain microvasculature of FcγRIIA^TGN/CD41-YFP mice injected with ICs. Thrombi were observed 5 min after IC injection (arrows), whereas brain vasculature leakage occurred after 10 min. (Scale bar: 50 μm.) null, FcyRIIAnull; TGN; FcyRIIATGN. Data are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001 using one-way ANOVA (A and D) and an unpaired t test (C, E, and F).

However, we suspected that other anatomical sites of platelet sequestration might exist, as the IVIS approach may not permit an optimal distinction between immobilized platelet aggregates and circulating platelets in the microvasculature, where small platelet aggregates are to be expected. In addition, if the entire platelet population was sequestered in the lungs, it was puzzling that none of the mice died from the IC trigger. Using a quantitative approach to measure fluorescently labeled CD41⁺ platelets, we estimated that the lungs, in fact, contained only 16% of the total platelet load (Fig. 4E), confirming that other sites of platelet sequestration likely existed.

The presence of platelets outside the vasculature was excluded first, as no platelets were detected in the thoracic lymph (Fig. S6A). Furthermore, assessment using whole-mouse imaging of yellow fluorescent protein (YFP) in FcγRIIA^TGN/CD41-YFP mice, which constitutively express YFP in CD41-expressing cells, efficiently identified thrombi in the lungs and megakaryocytes in the bone marrow, but did not reveal platelets outside blood vessels or in other tissues (Fig. S6B). Therefore, we speculated that tracking ICs in mice might be a more efficient means of leading us to platelet sequestration sites. We found that ICs not only localized to the lungs but were also observed in the brain vasculature (Fig. 4F). Thus, the brain microvasculature was examined using two-photon microscopy in live FcγRIIA^TGN/CD41-YFP mice, with the inclusion of FcγRIIA^null/CD41-YFP mice for comparison. We observed profound leakage of the brain vasculature in both FcγRIIA^null and FcγRIIA^TGN mice when injected with ICs (Fig. 4G and Movies S2 and S3). Of importance, in the presence of ICs, small platelet aggregates readily formed in the leaky brain microvasculature, but only if FcγRIIA was expressed by platelets (Fig. 4G and Movie S2). Thrombi were not detected in the microvasculature of the kidney, liver, or spleen of FcγRIIA^TGN mice injected with diluent or ICs (Fig. S6C), and two-photon microscopy of the femurs in live FcγRIIA^TGN/CD41-YFP mice did not reveal any platelet aggregation in sinusoids in the bone marrow (Fig. S6D). These data suggest that in the presence of ICs, platelets sequester in certain microvascular beds, notably in the lungs and brain, and that this event occurs independent of thrombosis and leakage.

Role of Platelet FcγRIIA and Serotonin in Acute Inflammatory Responses.

During microbial invasion, foreign antigens are recognized by host antibodies and form ICs. Hence, incubation of human platelets in the presence of influenza virus or various strains of bacteria leads to platelet activation, which strictly requires the presence of plasma and FcγRIIA (17, 18). While these observations suggest that motifs on pathogens contribute to the formation of ICs, whether they can trigger platelet activation in vivo has not been established.

Antibodies against lipopolysaccharide (LPS), a common gram-negative pathogen-associated molecular pattern (PAMP), were detected in the blood of healthy volunteers similar to patients with septic shock due to confirmed gram-negative bacteria infection (Fig. 5A). In contrast to humans, mice housed in a facility with high standards of cleanliness present no detectable anti-LPS antibodies (nonimmune mice) (Fig. 5B). Therefore, we immunized mice with small quantities of bacterial PAMP (LPS), which had no perceptible effect on FcγRIIA^null or FcγRIIA^TGN mice (Fig. 5C). FcγRIIA^null and FcγRIIA^TGN mice developed equivalent anti-LPS antibody levels within 3 wk (LPS-immune mice) (Fig. 5B), and, importantly, a subsequent injection of LPS only induced serotonin release and shock in FcγRIIA^TGN mice (Fig. 5 C and D). The shock induced by LPS-containing ICs was dependent on the presence of platelets, and was greatly diminished in the absence of peripheral serotonin or β3 gene expression (Fig. 5 E–G). Moreover, plasma serotonin levels were significantly reduced in FcγRIIA^TGN/β3^−/− mice after LPS injection (Fig. 5D). Thrombocytopenia was dramatically increased in the presence of FcγRIIA expression (Fig. 5H), as in the passive model of the IC trigger, and was also transient, as platelets with significantly reduced PF4 and serotonin content were identified in blood when they reappeared (Fig. 5I).

Fig. 5.

Role of the FcγRIIA/serotonin axis in acute inflammation. Endogenous antibodies directed against LPS (anti-LPS) were measured by ELISA in plasma of healthy volunteers (HV) and patients with ongoing septic shock (SS) (A; n = 11) and in FcγRIIA^null and FcγRIIA^TGN mice that were immunized with LPS (LPS-immune) or diluent (nonimmune) (B; n = 4). OD, optical density. (C) Systemic shock and temperature were evaluated in nonimmune (n = 4) or LPS-immune mice (n = 6) expressing or not expressing FcγRIIA. (D) Serotonin levels in plasma were determined in LPS-immune FcγRIIA^null, FcγRIIA^TGN, and FcγRIIA^TGN/β3^−/− mice immediately after LPS injection (n = 5) and compared with nonimmune FcγRIIA^TGN mice (baseline level, indicated as a dotted line). Systemic shock and temperature were measured after LPS injection in LPS-immune FcγRIIA^TGN mice preinjected with platelet-depleting (PLT Ab) or control (Ctrl Ab) antibodies (E; n = 5) and in LPS-immune FcγRIIA^TGN/Tph1^+/+ mice and FcγRIIA^TGN/Tph1^−/− mice (F; n = 6). (G) Systemic shock was measured after LPS injection in LPS-immune FcγRIIA^TGN/β3^+/+ mice and FcγRIIA^TGN/β3^−/− mice (n = 4). (H) CD41⁺ platelets in whole blood were counted using flow cytometry at the indicated time points after injection of LPS in LPS-immune FcγRIIA^null and FcγRIIA^TGN mice (n = 5). (I) Platelet content in PF4 (n = 5; Left) and serotonin (n = 6; Right) were determined 48 h after LPS injection in LPS-immune FcγRIIA^null and FcγRIIA^TGN mice and compared with nonimmune FcγRIIA^TGN mouse levels (dotted lines). Dil, diluent; null, FcyRIIAnull; TGN, FcyRIIATGN. Data are mean ± SEM. **P < 0.005, ***P < 0.001, and ****P < 0.0001, using an unpaired t test (A and I); repeated-measures two-way ANOVA, statistical variation between groups (C and E–G); and one-way ANOVA (B, D, and H).

We further verified whether the same conclusions hold in viremia and in a well-described model of active systemic anaphylaxis (23, 28, 29). The i.v. injection of herpes simplex virus-1 (HSV-1), capable of blood dissemination in immunized humans (45, 46), in HSV-1–immunized mice induced shock reminiscent of the responses seen in IC-injected mice and dependent on FcγRIIA (Fig. S7A). Moreover, BSA injection in BSA-immunized mice initiated systemic anaphylactic responses, and a proportion of the FcγRIIA^TGN mice, but never the FcγRIIA^null mice, died. Modest shock response and moderate thrombocytopenia were also observed in FcγRIIA^null mice (Fig. S7B), possibly due to the high antigenicity of BSA and the potent immunization protocol implicating adjuvant, consistent with other studies implicating neutrophils and FcγRIIA-independent responses in these experimental conditions (23, 28, 29). Of importance is that in all models, platelet FcγRIIA and serotonin were involved in shock, while FcγRIIA was implicated in thrombocytopenia (Fig. S7 C–E).

Thus, as in our passive model of IC-mediated immune reaction, active immunization with gram-negative PAMPs, virus, or protein antigen dominantly implicates platelet FcγRIIA and serotonin, and presents with transient platelet sequestration. The mechanisms unveiled in this study are illustrated in Fig. 6.

Fig. 6.

Platelets release pathogenic serotonin and return to blood circulation after IC-mediated degranulation and sequestration. Sequential events (numbered 1–6) were observed when circulating ICs encountered platelets. Platelets are abundant in blood and in humans (not in WT mice); they express FcγRIIA, a low-affinity receptor for IgG. ICs activate FcγRIIA present on platelets (1), which changes αIIbβ3 to its active conformation (2). (3) Active αIIbβ3 binds its extracellular ligand fibrinogen, which mediates outside-in signaling and granule release. In the absence of αIIbβ3, there is no granule release. It is suggested (dotted line) that serotonin engages neutrophils (4), and it is further hypothesized (dotted line) that serotonin induces vasodilatation through its action on endothelial cells (5). (6) Multiple manifestations are observed when ICs form in blood. Shock, characterized by loss of consciousness, immobility, shallow respiration, and hypothermia, strictly implicates platelets, αIIbβ3 binding to fibrinogen, and serotonin release. In the absence of neutrophils, serotonin is released but shock is abolished. It is surmised that neutrophils produce PAF in response to serotonin, which may contribute to shock downstream of serotonin release. Mediators of shock are indicated in the figure. Thrombocytopenia is due, at least in part, to platelet sequestration in certain vascular beds, notably in the lung and brain microvasculature. Sequestration implicates FcγRIIA but, in contrast to shock, occurs independent of serotonin and neutrophils, and only partially implicates β3. Thrombocytopenia is only transient, and platelets return to blood circulation with emptied granules. Microparticle release is observed in blood before return of platelets. Roles of different molecules in thrombocytopenia are indicated in the figure. Vasodilatation is implicating platelet-derived serotonin and is occurring independent of the presence of neutrophils. Thrombosis was characterized in lungs following IC administration. It implicates FcγRIIA and β3, and, in contrast to shock, it involves GPIb and not serotonin. These data support the notion that shock and thrombosis are independent events. Vascular leakage occurs independent of platelets and FcγRIIA. Molecules implicated in leakage are presented in the figure. NETosis, neutrophil extracellular traps; P-sel, P-selectin.

Discussion

In this study, we examined platelet activity in IC-induced systemic inflammation in the absence of vascular insult. We confirmed a central role for platelet activation and identified serotonin as a critical platelet component mediating mechanisms of shock.

Albeit artificial, models utilizing ferric chloride, laser-induced injury, or injection of cytokines or PAMP in tissue/organs (e.g., cremaster muscle, liver, lungs) (4 ⇓–6, 9, 47) have provided crucial insights to key pathways implicated in inflammation. Herein, we used HA-IgG as a surrogate model of ICs and revealed critical components in the in vivo response to systemic ICs, which were further confirmed using active immunization with endotoxin, virus, or a commonly used antigen in the study of anaphylaxis. Our observations were only possible using FcγRIIA^TGN mice, where we confirmed the functional association of FcγRIIA and αIIbβ3 in platelet activation, and suggest that targeting these receptors may have clinical benefits in severe conditions involving ICs. Conversely, other molecules (i.e., P-selectin, GPIb, ADP, thromboxane) that classically play a dominant role in platelet activation in thrombosis, or favor platelet and neutrophil interactions, were dispensable. The contribution of neutrophils to the systemic response to ICs is, however, not excluded, as neutrophils have also been previously implicated, possibly through the release of PAF (12, 23, 28). Hence, although serotonin release was maintained in the absence of neutrophils, shock was dramatically reduced (Figs. S2B and S8A). We thus propose that the release of serotonin precedes the neutrophil contribution to shock, consistent with the reported role of serotonin in neutrophil activation (48). As vasodilation was also present in the absence of neutrophils (Fig. S3B), these data suggest that platelets, through serotonin, orchestrate neutrophil activation and endothelial cell functions, and that these events can occur independently (Fig. 6).

Vascular leakage is another feature systematically observed when ICs were present in blood. Intriguingly, leakage and vasodilatation were not connected, as leakage took place independent of FcγRIIA but vasodilation critically required FcγRIIA and serotonin. As such, vessel permeability likely involved smaller postcapillaries vessels and more subtle changes in endothelial cell interactions, and was also insufficient to induce significant edema in the lungs, although platelets and neutrophils accumulated in great number in the lungs. In addition, leakage critically involved IC-mediated signaling, given that it was totally absent in FcRγ^−/− mice (Fig. S3A). While neutrophils express other IgG receptors (other than FcγRIIA), they were dispensable in the process (Fig. S3A). Therefore, leakage could be attributed to mast cells or basophils, for instance, which also express an array of receptors, such as FcγRI and FcγRIII, capable of responding to ICs and can mediate permeability, potentially through histamine release (49).

Whereas serotonin is mostly known for its role in mood, anxiety, psychosis, or memory in the central nervous system, more than 95% of total body serotonin is present in the periphery (39). The majority of peripheral serotonin is stored in platelets, and our observations further revealed that platelets from female mice contain less serotonin than those from males (Fig. S8B), which may explain, in part, the aggravated phenotype observed in male mice compared with females following IC challenge. Moreover, although platelets in females and males expressed similar levels of FcγRIIA, female FcγRIIA^TGN mice presented significantly lower platelet counts than males, which could also partially explain the reduced shock in females in comparison to male mice (Fig. S8 C–E). Other immune components, such as complement C5a, are also more abundant in males than in females (50). Whether the serotonin reservoir in platelets explains a fundamental gender-related dichotomy in susceptibility to inflammatory responses to ICs remains to be established.

The function of serotonin in platelets is not clear; studies suggest that it is important for the serotonylation of proteins necessary in platelet aggregation (51). However, SSRIs are typically used by patients during the perioperative period and mice lacking peripheral serotonin present only mild bleeding defects (43). Thus, the present study sheds light on a major role of platelet serotonin in response to a systemic stimulus, which occurs independent of other molecules typically implicated in the prevention of bleeding. The advantages for an organism to release bulk serotonin in response to systemic ICs are unclear. We can speculate that in response to a microbial invasion in an immune host, it might be preferable to reduce blood flow to prevent dissemination of the pathogen to vital organs and to facilitate its capture by phagocytes. As serotonin also mediates organ regeneration (52), its liberation may be pivotal to regrowth following insults caused by pathogen invasion.

Thrombocytopenia, which coincides with thrombi formation in the lungs, has been reported to occur in FcγRIIA^TGN mice, and requires the expression of guanine nucleotide exchange factor CalDAG-GEF1 and 12-lipoxygenase (22, 25 ⇓–27, 53). We showed here that the formation of thrombi or occlusion of blood vessels was not the primary cause of shock; thrombi formed normally in FcγRIIA^TGN/Tph1^−/− mice, whereas shock was nearly totally abrogated in these mice. Furthermore, reduction of thrombosis by blockade of GPIb was without effect on shock and thrombocytopenia, thereby revealing that thrombocytopenia resulted mainly from platelet sequestration, not thrombosis. Whereas platelets were observed in the lungs and brain, principally in the smaller and intertwined vessels, platelets may also be hiding in other yet-to-be-discovered vascular beds despite our careful investigations. In thrombotic thrombocytopenic purpura, profound thrombocytopenia is explained by the failure of ADAMTS13 to perform proteolysis of vWF attached to the endothelium (54), a mechanism distinct from what is observed in IC-induced thrombocytopenia, as the blockade of vWF and GPIb had no effects on thrombocytopenia in our study. As in-depth whole-mouse imaging uniquely identified megakaryocytes and thrombi, which comprise more stable and adherent platelets, it suggests that the majority of the sequestered platelets were dislodged by the perfusion procedure. Therefore, we propose that platelets might be bridged together by ICs, and that platelet–IC scaffolds may be loosely trapped in the microvasculature.

Degranulated platelets recirculate, a finding of potential significance for elucidating mechanisms underlying thrombocytopenia. The absence of PS at the surface suggests that they are not procoagulant platelets, known as balloon- or zombie-like platelets (55). How platelets return to the circulation after sequestration is unclear, but it is reasonable to speculate that disengagement of FcγRIIA after its desensitization by yet unknown mechanisms (e.g., unidentified immunoreceptor tyrosine-based inhibitory motif-containing receptors or phosphatases) or IC internalization by platelets (56) might release platelets from platelet–IC scaffolds and permit their liberation from the microvasculature. FcγRIIA expression was maintained, however, on recirculating platelets, suggesting that it might be recycled if the internalization of ICs is implicated. Of interest is that platelets at thrombi surfaces visualized in vascular injury models appear loosely packed and lightly activated (57, 58), and might also return to the circulation. As platelets activated in vitro with thrombin can also circulate after degranulation if transfused (59, 60), our study reveals that thrombotic and immunological triggers can induce degranulation independent of platelet elimination. These models contrast with the general belief that platelets “have only one life,” and may not recirculate after undergoing activation in vivo.

The insertion of human activating (FcγRIIA/IIIA/IIIB) and inhibitory (FcγRIIB) FcγR into the equivalent murine locus confirmed the predominance of FcγRIIA in systemic shock in the humanized mouse model (28), suggesting that our findings may well be translatable to humans as platelets from transgenic mice and humans express equivalent levels of FcγRIIA (18). While these approaches cannot fully recapitulate all of the subtleties of IC-driven inflammation in humans, it is very likely that the mechanisms revealed in our study may, at least in part, take place in disease states such as rheumatic disease, HIT, sepsis, viremia, anaphylaxis, and adverse reactions following i.v. IgG therapy.

PAMPs trigger innate immune responses through activation of pattern-recognition receptors, but the recognition of PAMPs by antibodies in adaptive immunity can modulate different responses (61). We found that LPS, a Toll-like receptor 4 (TLR-4) trigger, dominantly implicates platelets and serotonin when involving the adaptive immune response in mice that had been preexposed to LPS. All adult volunteers we examined also displayed antibodies directed against the endotoxin. Interestingly, humans lacking TLR signaling molecules are extremely susceptible to infections in infancy and childhood, and thereafter develop significant resistance, consistent with the prevalent role of adaptive immunity in adults (62). Sepsis susceptibility is associated with FcγRIIA polymorphism (63) and is accompanied by elevated serotonin and endothelial hyperpermeability (64), which could be attributed to platelets. Furthermore, thrombocytopenia measured in patients with sepsis is a strong predictor of mortality (65, 66), and our data suggest that platelets may be sequestered through FcγRIIA activation in some patients. It is thus important to note that most mouse models of sepsis or viral infection may be suboptimal, as they overlook the contribution of FcγRIIA in a nonimmune host. Observations from trials during which LPS was injected into volunteers revealed that humans responded more promptly to small doses of LPS than mice (67), pointing to the involvement of ICs and FcγRIIA in humans, a pathway absent in mice.

Circulating platelets presenting reduced granule content are reported in different contexts, such as cardiovascular diseases, type-2 diabetes mellitus, preeclampsia, autoimmune diseases, and sepsis (68 ⇓⇓⇓–72). In patients with severe sepsis, ADP and serotonin contents in platelets were reduced by 40% and 50%, respectively (71). However, very little is known about the impact of in vivo platelet degranulation on platelet life span. In chronic conditions implicating ICs, such as rheumatic diseases, ICs constantly trigger platelet FcγRIIA, consistent with the frequent shifts in platelet count. Hence, microthrombi are recognized in these diseases, including in the lungs, and the platelet content of serotonin is reportedly reduced in patients with SLE and RA by 25% and 27%, respectively (72, 73), suggesting that platelets might indeed circulate in their degranulated form in various contexts implicating ICs (70). Neuropsychiatric SLE is the least understood, yet the most prevalent, manifestation of SLE (74). Our identification of localized platelet activation in the brain microvasculature and leakage of the blood–brain barrier may thus have implications in the important and poorly understood neurological manifestations in rheumatic diseases.

In summary, our study reveals platelet contributions to inflammation in reactions involving ICs. It appears that the FcγRIIA signaling and serotonin release are unique in regard to their major role in inflammation and minor roles in the prevention of bleeding, suggesting that interference in this process might be a promising avenue for further research.

Materials and Methods

Mice.

C57BL/6J (FcγRIIA^null mice), FcγRIIA^TGN hemizygous mice (12, 20), and FcRγ^−/− mice were obtained from The Jackson Laboratory. FcγRIIA^TGN hemizygous mice described to express human FcγRIIA on platelets, megakaryocytes, monocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, and dendritic cells (12, 20) were backcrossed to C57BL/6J more than 10 times. The β3^−/− mice (30), Tph1^−/− mice (43), and CD41-YFP mice (75) were crossed with FcγRIIA^TGN mice to obtain FcγRIIA^TGN/β3^−/− (30), FcγRIIA^TGN/Tph1^−/−, and FcγRIIA^TGN/CD41-YFP mice. Chimeric mice were generated by transfer of bone marrow cells of FcγRIIA^TGN mice into FcγRIIA^null (WT), Fibγ^390-396A, and Fibγ^∆5 irradiated mice (34, 35, 76). Guidelines of the Canadian Council on Animal Care were followed in a protocol approved by the Animal Welfare Committee at Laval University (2013-106-3).

HA-IgG as an IC Model.

Human IgG (IC) (MP-biological, Sigma–Aldrich and Innovative Research) were i.v. injected (600 μg and 750 μg) in males and females, respectively. In some experiments, human IgG was dissolved but nonaggregated (monomeric) or mouse IgG was aggregated and injected in mice (29).

Measure of Shock.

Temperature was measured using a rectal probe thermometer at indicated time points. Signs of apparent shock were assessed as described previously (24, 77) using a score from 0 to 3 (24, 77). A score of 3 represents completely immobilized and unconscious mice (mice collapse and do not react to sound or touch), a score of 2 represents mice with impaired mobility and irregular respiration, a score of 1 corresponds to mice with slow motions and shallow respiration, and a score of 0 describes normal mice. In anaphylaxis experiments implicating BSA injection in BSA-immunized FcγRIIA^TGN mice, death was sometimes observed. In those exceptional cases, shock was scored as 4. Scores measured in experimental groups were averaged and are presented as a function of time (minutes). Scores for individual mice for key experiments are provided in SI Materials and Methods (Fig. S1 A and F).

LPS Immunization Model.

Mice were immunized with i.v. injection of 1 mg/kg LPS (Escherichia coli 0111:B4; Sigma–Aldrich) at days 0, 14, 28, and 42. At the first and the last immunizations, shock and temperature were monitored for 1 h. Mouse blood was drawn by cardiac puncture 10 min after the fourth injection and used for platelet count and ELISA. Lungs were also collected as described below.

Flow Cytometry.

Flow cytometry was performed using a BD FACSCanto II instrument with forward scatter coupled to a photomultiplier tube “small particles option” flow cytometer (BD Biosciences). Platelets, platelets interacting with neutrophils, and platelet microparticles were analyzed.

Histology.

In some experiments, organs were collected at the end of the experiment. Intratracheal instillation with 1 mL of 4% paraformaldehyde in lungs was performed before collection. Brain, kidneys, spleen, heart, liver, and lungs were collected and then fixed in 4% paraformaldehyde for 24 h (lungs) or 72 h (other organs); they were then washed and stored in PBS at 4 °C before histology. After fixation, paraffin-embedded organs were cut into 5-μm sections and stained with hematoxylin and eosin. Thrombi were observed on five different spots at 400× resolution, they were counted using light microscopy (BX51; Olympus) by a blinded investigator, and the numbers of lung thrombi per square millimeter were calculated. Neutrophils were identified in lungs of IC-injected FcγRIIA^TGN mice.

Two-Photon Intravital Microscopy.

For in vivo imaging of the mouse brain, FcγRIIA^TGN/CD41-YFP mice (8–12 wk old) were anesthetized with 1–2% isoflurane (vol/vol) and a cranial window was made to expose the vasculature of the sensorimotor cortex. Animals were imaged 2 wk after the surgery. Briefly, for the imaging session, the head of the mouse was restrained using a custom-built cranial stereotaxic apparatus (David Kopf Instruments) and placed under the microscope. For the ear imaging, mice were anesthetized and the hair recovering the ears was gently removed using Nair, a commercial depilatory lotion. One ear was then gently flattened and fixed on a Plexiglas bloc using MSI-EpiDermGlu (Medisav Services). Gelseal (GE Healthcare) was applied around the tissues to form a watertight rim, and the imaging cavities were filled with sterile HBSS without Ca²⁺/Mg²⁺ (Thermo Fisher Scientific). Blood vessels were stained by an i.v. injection of 0.05% Evans Blue (Sigma–Aldrich) diluted in 0.9% sterile saline. Body temperature was kept at 37 °C during all procedures with a heating pad. Four to six different vessels per mouse were analyzed at 1 and 8 min postinjection.

Patients with Septic Shock.

Adult (age ≥ 21 y) patients with septic shock and confirmed, gram-negative bacteremia were included. Each patient or an authorized family member provided written informed consent. Septic shock was defined as the presence of sepsis (requiring evidence of systemic infection and two or more of the following: temperature >38 °C or <36 °C; heart rate >90 beats per minute; respiratory rate >20 breaths per minute or partial pressure of carbon dioxide in arterial blood <32 mmHg; WBC count >12,000/mL, <4,000/mL, or >10% bands) and the need for vasopressors to maintain a systolic blood pressure >90 mmHg or within 40 mmHg of baseline despite adequate fluid resuscitation (5, 6). For identification of gram-negative bacteremia, blood samples were obtained from patients upon intensive care unit (ICU) admission as part of their routine clinical care. Blood samples underwent gram staining and culturing in a clinical pathology laboratory. Gram-negative pathogens were identified from the gram stain and/or cultures by the clinical laboratory. Healthy, fasting adult (age ≥ 21 y) control subjects provided informed consent. Following informed consent, demographic data, physiological parameters, and laboratory data were recorded. Plasma was harvested by centrifugation on whole blood collected in sterile ACD vacutainer tubes. Plasma was frozen at −80 °C until used for assays. In patients with septic shock, plasma was obtained within 48 h of ICU admission.

Study Approval.

Informed consent was obtained from all human subjects in the study. The study was approved by the institutional review board at University of Utah.

Statistical Analysis.

Results are presented as mean ± SEM. The statistical significance for comparisons between groups was determined using one-way ANOVA, two-way repeated- measures ANOVA, an unpaired Student’s t test, or a Mann–Whitney U test. The D’Agostino–Pearson test was used as a normality test. All statistical analysis was done using Prism software package 6 (GraphPad Software).

Acknowledgments

We thank Isabelle Dubuc for expert technical assistance with virus preparation. This work was supported by a foundation grant from the Canadian Institutes of Health Research (CIHR) (to E.B.); it was also supported, in part, by NIH/National Heart, Lung, and Blood Institute Grant 5F32HL118865 (to K.R.M.), Grant HL130054 (to P.J.N.), and Grant R01Hl68130 (to J.E.I.); by Grant 1K01DK111515 from the National Institutes of Diabetes and Digestive and Kidney Diseases (to K.R.M.); by a grant from the Canadian Funds for Innovation (to G.P.); by Grant HL112311 (to M.T.R.); by Grant HL126547 (to M.T.R.); by National Institute on Aging Grant AG048022 (to M.T.R.), by a CIHR foundation grant (to W.I.K.); by Grant P01HL110860 (to S.E.M.), by a CIHR operating grant (to S.L.); and by CIHR Grant MOP 93575 (to L.F.). E.B. is the recipient of an investigator award from the CIHR. K.R.M. and J.E.I. are American Society of Hematology Fellow Scholars. G.M. has an award from the Canadian Blood Services, N.T. and I.M. are recipients of fellowships from The Arthritis Society (TAS), A.Z. is a recipient of fellowships from the TAS and CIHR, and B.M. has a fellowship from the Fondation du Centre Hospitalier Universitaire de Quebec. This material is also the result of work supported with resources and the use of facilities at the George E. Wahlen Veterans Affairs Medical Center (M.T.R.). The contents do not represent the views of the US Department of Veterans Affairs or the US Government.

Footnotes

↵¹N.C. and I.A. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: eric.boilard{at}crchudequebec.ulaval.ca.

Author contributions: N.C., I.A., K.R.M., and E.B. designed research; N.C., I.A., G.M., K.R.M., B.M., A.Z., T.L., Y.B., N.T., I.M., and H.Z. performed research; G.P., M.T.R., J.E.I., L.F., S.E.M., F.C., B.N., W.I.K., M.J.F., P.J.N., S.L., and P.R.F. contributed new reagents/analytic tools; N.C., I.A., G.M., K.R.M., B.M., A.Z., and E.B. analyzed data; and N.C., I.A., and E.B. wrote the paper.
Conflict of interest statement: J.E.I. has a financial interest in and is a founder of Platelet BioGenesis, a company that aims to produce donor-independent human platelets from human-induced pluripotent stem cells at scale. J.E.I. is an inventor on this patent. The interests of J.E.I. were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720553115/-/DCSupplemental.

Published under the PNAS license.

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(2011) CalDAG-GEFI deficiency protects mice in a novel model of Fcγ RIIA-mediated thrombosis and thrombocytopenia. Blood 118:1113–1120.
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2. Krauel K
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(2009) Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 113:3716–3725.
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1. Zhi H, et al.
(2013) Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo. Blood 121:1858–1867.
OpenUrl Abstract/FREE Full Text
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1. Nieswandt B,
2. Varga-Szabo D,
3. Elvers M
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2. Ye S,
3. Whiteheart SW
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2. Delaney MK,
3. O’Brien KA,
4. Du X
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OpenUrl Abstract/FREE Full Text
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Proceedings of the National Academy of Sciences: 115 (7)

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Article Classifications

Biological Sciences
Immunology and Inflammation

[1] ↵
Semple JW,
Italiano JE Jr,
Freedman J
(2011) Platelets and the immune continuum. Nat Rev Immunol 11:264–274.
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[2] Semple JW,

[3] Italiano JE Jr,

[4] Freedman J

[5] ↵
Morrell CN,
Aggrey AA,
Chapman LM,
Modjeski KL
(2014) Emerging roles for platelets as immune and inflammatory cells. Blood 123:2759–2767.
OpenUrl Abstract/FREE Full Text

[6] Morrell CN,

[7] Aggrey AA,

[8] Chapman LM,

[9] Modjeski KL

[10] ↵
Kapur R,
Zufferey A,
Boilard E,
Semple JW
(2015) Nouvelle cuisine: Platelets served with inflammation. J Immunol 194:5579–5587.
OpenUrl Abstract/FREE Full Text

[11] Kapur R,

[12] Zufferey A,

[13] Boilard E,

[14] Semple JW

[15] ↵
Kolaczkowska E,
Kubes P
(2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13:159–175.
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[16] Kolaczkowska E,

[17] Kubes P

[18] ↵
Sreeramkumar V, et al.
(2014) Neutrophils scan for activated platelets to initiate inflammation. Science 346:1234–1238.
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[19] Sreeramkumar V, et al.

[20] ↵
Imhof BA, et al.
(2016) CCN1/CYR61-mediated meticulous patrolling by Ly6Clow monocytes fuels vascular inflammation. Proc Natl Acad Sci USA 113:E4847–E4856.
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[21] Imhof BA, et al.

[22] ↵
Goerge T, et al.
(2008) Inflammation induces hemorrhage in thrombocytopenia. Blood 111:4958–4964.
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[23] Goerge T, et al.

[24] ↵
Boulaftali Y, et al.
(2013) Platelet ITAM signaling is critical for vascular integrity in inflammation. J Clin Invest 123:908–916.
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[25] Boulaftali Y, et al.

[26] ↵
Gros A, et al.
(2015) Single platelets seal neutrophil-induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice. Blood 126:1017–1026.
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[27] Gros A, et al.

[28] ↵
Suurmond J,
Zou YR,
Kim SJ,
Diamond B
(2015) Therapeutics to block autoantibody initiation and propagation in systemic lupus erythematosus and rheumatoid arthritis. Sci Transl Med 7:280ps5.
OpenUrl FREE Full Text

[29] Suurmond J,

[30] Zou YR,

[31] Kim SJ,

[32] Diamond B

[33] ↵
Jönsson F, et al.
(2011) Mouse and human neutrophils induce anaphylaxis. J Clin Invest 121:1484–1496.
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[34] Jönsson F, et al.

[35] ↵
Bruhns P,
Jönsson F
(2015) Mouse and human FcR effector functions. Immunol Rev 268:25–51.
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[36] Bruhns P,

[37] Jönsson F

[38] ↵
Cai Z, et al.
(2015) Atomic description of the immune complex involved in heparin-induced thrombocytopenia. Nat Commun 6:8277.
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[39] Cai Z, et al.

[40] ↵
Pincetic A, et al.
(2014) Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol 15:707–716.
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[41] Pincetic A, et al.

[42] ↵
Gardiner EE,
Andrews RK
(2014) Structure and function of platelet receptors initiating blood clotting. Adv Exp Med Biol 844:263–275.
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[44] Andrews RK

[45] ↵
Bardina SV, et al.
(2017) Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity. Science 356:175–180.
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[46] Bardina SV, et al.

[47] ↵
Arman M, et al.
(2014) Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbβ3, and platelet factor 4. Blood 123:3166–3174.
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[48] Arman M, et al.

[49] ↵
Boilard E, et al.
(2014) Influenza virus H1N1 activates platelets through FcγRIIA signaling and thrombin generation. Blood 123:2854–2863.
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[50] Boilard E, et al.

[51] ↵
Duffau P, et al.
(2010) Platelet CD154 potentiates interferon-alpha secretion by plasmacytoid dendritic cells in systemic lupus erythematosus. Sci Transl Med 2:47ra63.
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[52] Duffau P, et al.

[53] ↵
McKenzie SE, et al.
(1999) The role of the human Fc receptor Fc gamma RIIA in the immune clearance of platelets: A transgenic mouse model. J Immunol 162:4311–4318.
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[54] McKenzie SE, et al.

[55] ↵
Ishii S, et al.
(1998) Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor. J Exp Med 187:1779–1788.
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[56] Ishii S, et al.

[57] ↵
Reilly MP, et al.
(2001) Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcγRIIA. Blood 98:2442–2447.
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[58] Reilly MP, et al.

[59] ↵
Jönsson F, et al.
(2012) Human FcγRIIA induces anaphylactic and allergic reactions. Blood 119:2533–2544.
OpenUrl Abstract/FREE Full Text

[60] Jönsson F, et al.

[61] ↵
Meyer T, et al.
(2015) CD32a antibodies induce thrombocytopenia and type II hypersensitivity reactions in FCGR2A mice. Blood 126:2230–2238.
OpenUrl Abstract/FREE Full Text

[62] Meyer T, et al.

[63] ↵
Stolla M, et al.
(2011) CalDAG-GEFI deficiency protects mice in a novel model of Fcγ RIIA-mediated thrombosis and thrombocytopenia. Blood 118:1113–1120.
OpenUrl Abstract/FREE Full Text

[64] Stolla M, et al.

[65] ↵
Amirkhosravi A, et al.
(2014) CalDAG-GEFI deficiency protects mice from FcγRIIa-mediated thrombotic thrombocytopenia induced by CD40L and beta2GPI immune complexes. J Thromb Haemost 12:2113–2119.
OpenUrl CrossRef PubMed

[66] Amirkhosravi A, et al.

[67] ↵
Arman M,
Krauel K
(2015) Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis. J Thromb Haemost 13:893–908.
OpenUrl CrossRef PubMed

[68] Arman M,

[69] Krauel K

[70] ↵
Gillis CM, et al.
(2016) Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice. J Allergy Clin Immunol 139:1253–1265.e14.
OpenUrl

[71] Gillis CM, et al.

[72] ↵
Bruhns P, et al.
(2009) Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 113:3716–3725.
OpenUrl Abstract/FREE Full Text

[73] Bruhns P, et al.

[74] ↵
Zhi H, et al.
(2013) Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo. Blood 121:1858–1867.
OpenUrl Abstract/FREE Full Text

[75] Zhi H, et al.

[76] ↵
Nieswandt B,
Varga-Szabo D,
Elvers M
(2009) Integrins in platelet activation. J Thromb Haemost 7(Suppl 1):206–209.
OpenUrl PubMed

[77] Nieswandt B,

[78] Varga-Szabo D,

[79] Elvers M

[80] ↵
Ren Q,
Ye S,
Whiteheart SW
(2008) The platelet release reaction: Just when you thought platelet secretion was simple. Curr Opin Hematol 15:537–541.
OpenUrl CrossRef PubMed

[81] Ren Q,

[82] Ye S,

[83] Whiteheart SW

[84] ↵
Li Z,
Delaney MK,
O’Brien KA,
Du X
(2010) Signaling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 30:2341–2349.
OpenUrl Abstract/FREE Full Text

[85] Li Z,

[86] Delaney MK,

[87] O’Brien KA,

[88] Du X

[89] ↵
Flick MJ, et al.
(2007) Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin alphaMbeta2 binding motif. J Clin Invest 117:3224–3235.
OpenUrl CrossRef PubMed

[90] Flick MJ, et al.

[91] ↵
Flick MJ, et al.
(2004) Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo. J Clin Invest 113:1596–1606.
OpenUrl CrossRef PubMed

[92] Flick MJ, et al.

[93] ↵
Ehlers R, et al.
(2003) Targeting platelet-leukocyte interactions: Identification of the integrin Mac-1 binding site for the platelet counter receptor glycoprotein Ibalpha. J Exp Med 198:1077–1088.
OpenUrl Abstract/FREE Full Text

[94] Ehlers R, et al.

[95] ↵
Kato K, et al.
(2004) Genetic deletion of mouse platelet glycoprotein Ibbeta produces a Bernard-Soulier phenotype with increased alpha-granule size. Blood 104:2339–2344.
OpenUrl Abstract/FREE Full Text

[96] Kato K, et al.

[97] ↵
Shrimpton CN, et al.
(2002) Localization of the adhesion receptor glycoprotein Ib-IX-V complex to lipid rafts is required for platelet adhesion and activation. J Exp Med 196:1057–1066.
OpenUrl Abstract/FREE Full Text

[98] Shrimpton CN, et al.

[99] ↵
Vanhoutte PM
(2016) Regenerated endothelium and its senescent response to aggregating platelets. Circ J 80:783–790.
OpenUrl CrossRef PubMed

[100] Vanhoutte PM

[101] ↵
Adnot S,
Houssaini A,
Abid S,
Marcos E,
Amsellem V
(2013) Serotonin transporter and serotonin receptors. Handb Exp Pharmacol 218:365–380.
OpenUrl

[102] Adnot S,

[103] Houssaini A,

[104] Abid S,

[105] Marcos E,

[106] Amsellem V

[107] ↵
Cloutier N, et al.
(2012) Platelets can enhance vascular permeability. Blood 120:1334–1343.
OpenUrl Abstract/FREE Full Text

[108] Cloutier N, et al.

[109] ↵
Zhang X,
Beaulieu JM,
Sotnikova TD,
Gainetdinov RR,
Caron MG
(2004) Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217.
OpenUrl Abstract/FREE Full Text

[110] Zhang X,

[111] Beaulieu JM,

[112] Sotnikova TD,

[113] Gainetdinov RR,

[114] Caron MG

[115] ↵
Côté F, et al.
(2003) Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc Natl Acad Sci USA 100:13525–13530.
OpenUrl Abstract/FREE Full Text

[116] Côté F, et al.

[117] ↵
Fuchs TA, et al.
(2010) Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA 107:15880–15885.
OpenUrl Abstract/FREE Full Text

[118] Fuchs TA, et al.

[119] ↵
Berrington WR, et al.
(2009) Clinical correlates of herpes simplex virus viremia among hospitalized adults. Clin Infect Dis 49:1295–1301.
OpenUrl PubMed

[120] Berrington WR, et al.

[121] ↵
Zahariadis G,
Jerome KR,
Corey L
(2003) Herpes simplex virus-associated sepsis in a previously infected immunocompetent adult. Ann Intern Med 139:153–154.
OpenUrl PubMed

[122] Zahariadis G,

[123] Jerome KR,

[124] Corey L

[125] ↵
McDonald B, et al.
(2010) Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330:362–366.
OpenUrl Abstract/FREE Full Text

[126] McDonald B, et al.

[127] ↵
Duerschmied D, et al.
(2013) Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice. Blood 121:1008–1015.
OpenUrl Abstract/FREE Full Text

[128] Duerschmied D, et al.

[129] ↵
Binstadt BA, et al.
(2006) Particularities of the vasculature can promote the organ specificity of autoimmune attack. Nat Immunol 7:284–292.
OpenUrl CrossRef PubMed

[130] Binstadt BA, et al.

[131] ↵
Strait RT, et al.
(2011) MHC class I-specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice. J Exp Med 208:2525–2544.
OpenUrl Abstract/FREE Full Text

[132] Strait RT, et al.

[133] ↵
Walther DJ, et al.
(2003) Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release. Cell 115:851–862.
OpenUrl CrossRef PubMed

[134] Walther DJ, et al.

[135] ↵
Lesurtel M, et al.
(2006) Platelet-derived serotonin mediates liver regeneration. Science 312:104–107.
OpenUrl Abstract/FREE Full Text

[136] Lesurtel M, et al.

[137] ↵
Yeung J, et al.
(2014) Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation. Blood 124:2271–2279.
OpenUrl Abstract/FREE Full Text

[138] Yeung J, et al.

[139] ↵
Dong JF, et al.
(2002) ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood 100:4033–4039.
OpenUrl Abstract/FREE Full Text

[140] Dong JF, et al.

[141] ↵
Podoplelova NA, et al.
(2016) Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting. Blood 128:1745–1755.
OpenUrl Abstract/FREE Full Text

[142] Podoplelova NA, et al.

[143] ↵
White JG,
Krumwiede MD,
Cocking-Johnson DJ,
Escolar G
(1996) Uptake of vWF-anti-vWF complexes by platelets in suspension. Arterioscler Thromb Vasc Biol 16:868–877.
OpenUrl Abstract/FREE Full Text

[144] White JG,

[145] Krumwiede MD,

[146] Cocking-Johnson DJ,

[147] Escolar G

[148] ↵
Stalker TJ, et al.
(2013) Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood 121:1875–1885.
OpenUrl Abstract/FREE Full Text

[149] Stalker TJ, et al.

[150] ↵
Welsh JD, et al.
(2016) A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature. Blood 127:1598–1605.
OpenUrl Abstract/FREE Full Text

[151] Welsh JD, et al.

[152] ↵
Michelson AD, et al.
(1996) In vivo tracking of platelets: Circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci USA 93:11877–11882.
OpenUrl Abstract/FREE Full Text

[153] Michelson AD, et al.

[154] ↵
Berger G,
Hartwell DW,
Wagner DD
(1998) P-Selectin and platelet clearance. Blood 92:4446–4452.
OpenUrl Abstract/FREE Full Text

[155] Berger G,

[156] Hartwell DW,

[157] Wagner DD

[158] ↵
Kawai T,
Akira S
(2010) The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors. Nat Immunol 11:373–384.
OpenUrl CrossRef PubMed

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Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration

Significance

Abstract

Results

Platelets Are Critical During the Systemic Response.

Identification of the Shock Mediator.

And the Platelet Count?

Localization of Sequestration Sites.

Role of Platelet FcγRIIA and Serotonin in Acute Inflammatory Responses.

Discussion

Materials and Methods

Mice.

HA-IgG as an IC Model.

Measure of Shock.

LPS Immunization Model.

Flow Cytometry.

Histology.

Two-Photon Intravital Microscopy.

Patients with Septic Shock.

Study Approval.

Statistical Analysis.

Acknowledgments

Footnotes

References

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Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration

Significance

Abstract

Results

Platelets Are Critical During the Systemic Response.

Identification of the Shock Mediator.

And the Platelet Count?

Localization of Sequestration Sites.

Role of Platelet FcγRIIA and Serotonin in Acute Inflammatory Responses.

Discussion

Materials and Methods

Mice.

HA-IgG as an IC Model.

Measure of Shock.

LPS Immunization Model.

Flow Cytometry.

Histology.

Two-Photon Intravital Microscopy.

Patients with Septic Shock.

Study Approval.

Statistical Analysis.

Acknowledgments

Footnotes

References

Citation Manager Formats

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