Reduction of mortality, cardiac damage, and cerebral damage by IL-1 inhibition in a murine model of TTP Free

Immune-mediated thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening thrombotic microangiopathy caused by anti-ADAMTS13 autoantibodies and severe deficiency of ADAMTS13, the enzyme involved in cleavage of prothrombotic von Willebrand factor (VWF) multimers.¹ The disease is characterized by formation of systemic microthrombi, resulting from a 2-step mechanism, namely inhibition of ADAMTS13, and endothelial degranulation leading to exocytosis of ultralarge VWF multimers.² TTP is characterized by severe thrombocytopenia, hemolytic anemia, and organ ischemia, mainly affecting the heart and brain. Despite numerous treatments, including plasma exchanges, corticosteroids, rituximab, and more recently, caplacizumab, TTP remains a fatal disease for ∼5% of patients,³ with cardiac ischemia representing the primary cause of death.⁴ Cardiac and cerebral ischemia may also be associated with long-term sequelae.^5,6 

The interleukin-1 (IL-1) pathway is known to drive sterile inflammation after ischemia.⁷ This pathway relies on 2 cytokines, IL-1α and -1β, which bind to the same signaling receptor (interleukin 1 receptor, type 1 [IL-1R1]). IL-1α behaves as a damage-associated molecular pattern (DAMP) because it is constitutively present in the cytoplasm of cells and can be released into the extracellular compartment after ischemia-induced cell death.⁸ IL-1β is mainly produced by leukocytes, and its transcription and processing may be activated by various DAMPs released during cell death. These 2 cytokines, which are released after ischemia, contribute significantly to tissue damage.^9-12 The size of experimental infarcts is reduced in IL-1α and IL-1β knockout (KO) mice as well as in IL-1R1 KO mice.^13-15 Anakinra, a recombinant form of the natural IL-1R antagonist, inhibits both biological effects of IL-1α and IL-1β and thus reduces both infarct size and postischemic impaired cardiac function in various in vivo studies.^16-18 Anakinra as an inhibitor of the IL-1 signaling pathway is therefore a promising target for the treatment of organ ischemia, which has recently been investigated in human clinical trials during acute coronary syndrome.^19-22 

TTP is an ischemic disease in which the involvement of IL-1 pathway has not been well demonstrated to date. Only 1 study reported slightly higher levels of IL-1β at diagnosis than during remission in 13 patients.²³ Given recent reports demonstrating the key function of IL-1 in ischemia,^24,25 we aimed to study the implication of IL-1 pathway in cardiac and brain ischemia–induced injury during TTP.

First, we evaluated activation of the IL-1 pathway in patients with TTP based on plasma levels of circulating IL-1α and -1β. Then, we inhibited IL-1 with anakinra in a severe mouse model of TTP. Finally, we tested the ability of IL-1 to trigger endothelial degranulation and thus VWF release, a critical step in TTP pathogenesis.

A prospective study was conducted in France between 2008 and 2011, which consisted of a National Clinical Research Project (no. 2007/23) approved by the ethical committee of the Assistance Publique-Hôpitaux de Marseille, and after informed consent according to the Declaration of Helsinki. Diagnostic criteria for TTP and then for patient classification (survivor and nonsurvivor) are described in supplemental Data, available on the Blood website. For each patient in the acute phase, platelet-poor plasma was obtained from the first therapeutic plasma exchange and thus before treatment of the disease. Plasma from the first plasma exchange of patients with autoimmune neurological diseases (myasthenia gravis or polyradiculoneuritis) formed the negative controls. The control patients therefore have 2 decisive characteristics in common with the patients with TTP: an autoimmune disease and a plasma collection that was carried out at the time of the first plasma exchange. Plasma from patients with TTP was also collected during remission by venipuncture into sodium citrate.

Plasma IL-1α concentrations from patients with TTP and control patients were measured by enzyme-linked immunosorbent assay (ELISA, IL-1α human ELISA kit, Invitrogen). IL-1β levels in plasma from patients with TTP and control patients were determined by ProQuantum High-Sensitivity Immunoassays (IL-1β human ProQuantum Immunoassay Kit, Thermo Fisher). Troponin T and PS-100b concentrations were measured in TTP plasma by the electrochemiluminescence technique (Cobas 8000, module e602, Roche).

Male and female ADAMTS13 KO mice (B6.129-ADAMTS13tm1Dgi²⁶) on C57Bl6xCASA background (with elevated VWF plasma levels) were kept and raised in our facilities on a 12-hour light/dark cycle, with free access to food and water. At the time of the experiments, the mice were 12 to 15 weeks old and had a body weight of 18 to 26 g. The mice were examined clinically on a daily basis, paying particular attention to their general condition, weight, and stress levels. All experiments were performed in accordance with National Institutes of Health guidelines and were approved by the ethics committee in charge of animal experimentation of our institution (national agreement: D1305520, APAFIS#30226-2021050614583963).

A murine model of VWF-induced TTP was driven in ADAMTS13 KO mice by intravenous injection of 1500 IU/kg body weight of recombinant human VWF in the retro-orbital sinus (Veyvondi, Takeda) on days 0, 1, and 2. Control mice received daily retro-orbital injections of 0.9% NaCl of equivalent volume (vehicle). Treated mice received daily intraperitoneal injection of anakinra (Sobi) at 100 mg/kg from day 0 to day 5, whereas untreated mice received intraperitoneal injection of 0.9% NaCl (placebo) of equivalent volume. Anakinra or placebo was injected 5 minutes after VWF on days 0, 1, and 2. Four groups of mice were thus constituted: “control” (injection of vehicle and placebo), “anakinra” (injection of vehicle and anakinra), “TTP” (injection of VWF and placebo), and “TTP + anakinra” (injection of VWF and anakinra).

To test the efficacy of the dosage of anakinra at 100 mg/kg, 2 protocols have been designed. The first included blood samples at day 3 and a survival study up to day 6 (protocol no. 1: survival study; supplemental Figure 1A). The second included imaging (ultrasound and isotope imaging) at day 0 and day 2 before cervical dislocation at day 2 and a histologic study (protocol no. 2: isotope imaging and histologic studies; supplemental Figure 1B). Each outcome was analyzed by a 1-way analysis of variance on rank (the Kruskal-Wallis test), then in case of P value < .05, by a further comparison of each group to the reference group (TTP) performing Dunn correction.

A murine model of IL-1–induced TTP was driven in ADAMTS13 KO and wild-type (WT) mice by intraperitoneal injections of 10 ng/g human recombinant IL-1α or IL-1β (Bio-Techne) every hour for 9 hours (supplemental Figure 1C).

Blood samples were collected from the facial vein of mice anesthetized with 1% to 2% isoflurane. Hematocrit, hemoglobin, and platelet counts were performed on EDTA-treated blood diluted to 1:3000 using a veterinary hematology analyzer (Leytemed). Blood smear was stained with May-Grünwald Giemsa stain for manual assessment of schistocytes, and the remaining blood was centrifuged at 2000g for 15 minutes for plasma extraction. Troponin I levels in plasma from TTP and control mice were determined by ELISA (high sensitivity mouse cardiac troponin-I ELISA, Life Diagnostic). IL-1α and -1β concentrations in murine plasma were determined by ELISA (IL-1α ELISA kit, Invitrogen; and IL-1β ProQuantum Immunoassay Kit, Thermo Fisher, respectively). PS-100b levels were determined by ELISA (Mouse S100B ELISA Kit, MyBioSource). D-dimer concentrations were determined by ELISA (Mouse D-2D, MyBioSource). VWF antigen levels were measured in mouse plasma with an in-house ELISA, essentially as previously described, using a pair of polyclonal rabbit anti-VWF antibodies (Agilent Technologies).²⁷ Normal citrated pooled plasma obtained from WT C57B6 mice was used as reference. VWF multimer profile was performed, essentially as previously described, in 2% sodium dodecyl sulfate–agarose gels.²⁸ VWF was detected with an in-house alkaline phosphatase–conjugated polyclonal anti-VWF and colorimetric alkaline phosphatase-substrate kit (Bio-Rad Laboratories). Membranes were imaged with a G:BOX Chemi XT16 Image Systems (Syngene). Multimer profiles were analyzed using the Gel Analyzer tool of ImageJ. High-molecular-weight multimers were defined as all peaks beyond the 10th peak.

In vivo heart structure and function were evaluated at day 0 and day 2 using a high-frequency scanner (Vevo2100 VisualSonics). Briefly, mice were anesthetized with 1% to 2% isoflurane inhalation and placed on a heated platform to maintain temperature during the analysis. Two-dimensional imaging was recorded with a 22 to 55 MHz transducer (MS550D) to capture long- and short-axis projections with guided M-Mode and B-Mode and analysis with VevoLab software (VisualSonics). A target heart rate of 450 ± 100 beats per minute was used to record the M-mode. Ejection fraction was defined as the difference between the telediastolic and telesystolic volume, divided by the telediastolic volume. Shortening fraction was defined as the difference between the end-diastolic and telesystolic diameters, divided by the end-diastolic diameter.

Micro–positron emission tomography (PET)/computed tomography acquisitions were performed at day 0 and day 2 on a Nanoscan PET-computed tomography camera (Mediso). [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) tracer was injected intraperitoneally. Mice were maintained under 1% to 2% isoflurane anesthesia during acquisition. Static PET imaging was performed 1 hour after radiotracer injection, over 20 minutes. Quantitative region-of-interest analysis of the PET signal was performed using Invicro VivoQuant 4.0 software (Mediso), and tissue uptake values were expressed as a mean percentage of the injected dose per gram of tissue (%ID/g) for [¹⁸F]FDG. [¹⁸F]FDG was purchased as a ready-to-use radiopharmaceutical (Gluscan, Advanced Accelerator Applications).

Methods for heart histology are described in supplemental Methods.

Methods for brain imaging and brain histology and in vitro endothelial cell studies are described in supplemental Methods.

Methods for statistical analysis are described in supplemental Methods.

All experiments in humans were conducted within the framework of a national clinical research project approved by the ethics committee of Assistance Publique-Hôpitaux de Marseille (no. 2007/23). All experiments in mice were carried out in accordance with National Institutes of Health guidelines and were approved by our institution's animal experimentation ethics committee (national agreement: D1305520, APAFIS#30226-2021050614583963).

In total, 30 plasmas from patients with acute phase TTP (20 survivors and 10 nonsurvivors), 10 plasmas from control patients, and 10 plasmas from patients with TTP in remission were first analyzed (detailed information of TTP and control patients in Table 1). IL-1α and -1β concentrations were significantly higher in nonsurviving patients with TTP than in survivors (P = .044; Figure 1A-B). IL-1α and -1β concentrations were significantly higher in the surviving patients with TTP than in the control group (P = .028 and P < .001, respectively; Figure 1A-B). In patients tested during remission, plasma IL-1α and IL-1β concentrations were significantly lower than those observed in the acute phase of the disease (P = .006 and P = .004, respectively; Figure 1A-B). IL-1α and -1β plasma concentrations correlated during the acute phase (r² = 0.692, moderate correlation, P < .001; supplemental Figure 2A). The ratio of IL-1α to IL-1β was significantly higher in patients with TTP who did not survive than in survivors (P = .002; supplemental Figure 2B).

Ischemic damages in TTP primarily affect the heart and brain. We retrospectively assessed involvement of these 2 major organs by measuring troponin T and PS-100b concentrations in plasma of patients with TTP. Plasma troponin levels were significantly higher in nonsurvivors than in survivors (P = .031; supplemental Figure 2C), as were PS-100b levels (P = .022; supplemental Figure 2D). Plasma troponin and PS-100b concentrations strongly correlated with IL-1α and -1β concentrations in patients with TTP (troponin and IL-1α: r² = 0.714, strong correlation, P < .0001; troponin and IL-1β: r² = 0.758, strong correlation, P < .0001; PS-100b and IL-1α: r² = 0.538, moderate correlation, P < .0001; and PS-100b and IL-1β: r² = 0.546, moderate correlation, P < .001; Figure 1C-F). We found no correlation between IL-1α and IL-1β plasma levels and anti-ADAMTS13 immunoglobulin concentrations in patients with TTP (P = .85 and P = .54, respectively; r² = 0.03 and r² = 0.12, weak correlation for both; supplemental Figure 2E-F).

Based on these preliminary observations, we asked whether IL-1 may be involved in a TTP murine model. We first explored a model of TTP using daily intravenous injection of VWF (1500 IU/kg) for 3 days in ADAMTS13 KO mice. This protocol (supplemental Figure 1A-B) induced acute TTP characterized by severe thrombocytopenia, mechanical hemolytic anemia (schistocytes), and capillary microthrombi (supplemental Figure 3A-E). This protocol showed high mortality rate, with 70% of mice having died at day 4 (supplemental Figure 3F). Plasma IL-1α and IL-1β concentrations were significantly elevated (supplemental Figure 3G-H).

We then assessed the efficacy of anakinra in this murine model. We first determined the dose of anakinra to study, based on dose-response evaluation. TTP was induced in 36 mice randomly assigned to 6 groups treated with 1, 10, 25, 50, 100, and 200 mg/kg per day anakinra, respectively (Figure 2A). Plasma troponin and PS-100b concentrations were measured and compared at the peak disease severity (day 3). The 100-mg/kg dose was the lowest dose to achieve a statistically effect for both changes in plasma troponin and PS-100b levels (Figure 2B-C). The lowest median survival rate was observed in the group with the lowest dose (1 mg/kg), whereas the 2 highest doses (100 and 200 mg/kg) had the highest survival rate, but these results were not statistically significant (P = .07; Figure 2D). We therefore selected the 100 mg/kg dose of anakinra.

A survival study was then conducted on male mice divided into 4 groups: with or without TTP, and with or without treatment with 100 mg/kg anakinra per day (protocol no. 1; supplemental Figure 1A). TTP induced a significant excess mortality compared with the 2 control groups, which was significantly reduced by the administration of anakinra (P = .004; Figure 2E).

To further understand underlying mechanisms of beneficial effect of anakinra on mortality, additional male mice were included to evaluate specific cardiac and brain damages (protocol no. 2; supplemental Figure 1B).

Cardiac involvement was assessed using a multimodality approach, including troponin assays, echocardiography, PET imaging, and histology. TTP mice exhibited a significant increase of troponin concentrations compared with control mice (P < .001; Figure 3A), which were significantly reduced by anakinra treatment (P = .038). TTP also resulted in significant alterations in shortening fraction compared with that of controls (P < .001), which were significantly reduced by anakinra (P = .035; Figure 3B-C). Moreover, TTP induced a significant increase of [¹⁸F]FDG cardiac uptake compared with controls (P < .001; Figure 3D-E), which was significantly reduced by anakinra (P < .001). Evaluation of ischemic myocardial lesions based on histological scale was consistent with previous assessments: significant histological lesions in the TTP group compared with the control group (P = .003; Figure 3F-G) were reduced by anakinra (P = .043).

Evaluation of brain involvement included analysis of PS-100b levels, [¹⁸F]FDG brain and technetium-99m–coupled diethylene-triamine-penta-acetic acid ([^99mTc]Tc-DTPA) uptake, and histological assessment. Mice from the TTP group exhibited a significantly higher increase of PS-100b plasma levels than the control group (P = .016; Figure 4A), which was significantly reduced by anakinra treatment (P = .041). TTP also resulted in significant increase in [¹⁸F]FDG brain uptake compared with controls (P = .036; Figure 4B-C), which was significantly reduced by anakinra (P = .023). Moreover, TTP was associated with significant [^99mTc]Tc-DTPA brain uptake increase compared with control (P < .001; Figure 4D-E), which was significantly reduced by anakinra (P = .037). Analysis of brain tissue from mice showed that TTP caused significant neuronal damage compared with controls (P < .001; Figure 4F-G), which was significantly reduced by anakinra (P < .001).

During TTP, thrombocytopenia is known to be the result of platelet aggregation on high-molecular-weight VWF multimers previously degranulated by endothelial cells. Then, microthrombi resulting from this process are responsible for mechanical red blood cell lysis and anemia. Hematological parameters (anemia and thrombopenia) are therefore indirect markers of the occurrence of capillary microthrombi. Unexpectedly, in our TTP murine model, we observed that anakinra significantly improved the number of red blood cells (P = .029; supplemental Figure 4A) and platelets (P = .035; supplemental Figure 4B). Analysis of myocardial capillary microthrombi by immunofluorescence on myocardial sections from 18 hearts of mice with or without TTP, treated or not with anakinra (supplemental Figure 4C-D), demonstrated that hearts of treated mice had fewer capillary microthrombi than those of nontreated mice (P = .008).

Given the decrease in the number of capillary microthrombi in mice treated with an IL-1 receptor antagonist, and because preliminary studies show no effect of anakinra on VWF reactivity (supplemental Figure 5A-E), we wondered whether IL-1 itself is involved in endothelial cell degranulation. We then stimulated human cardiac microvascular endothelial cells (HMVECs) with phosphate-buffered saline (PBS), IL-1α, or IL-1β for 1 hour and observed significantly higher VWF concentrations in IL-1α– and -1β–stimulated cell supernatants (P = .008 and P = .008 for IL-1α and -1β, respectively; Figure 5A). IL-1α– and -1β–induced VWF release was detectable as early as 15 minutes and did not change significantly after 30 minutes of stimulation, in favor of a rapid VWF release mechanism (Figure 5B-C). The rapid release of VWF by endothelial cells is known to be calcium dependent. We then blocked intracellular calcium signalization with MAPTAM, a cell-permeable calcium chelator. MAPTAM significantly decreased IL-1–induced VWF release (IL-1α: P = .036, and IL-1β: P = .036; Figure 5A), confirming that IL-1–induced endothelial degranulation was calcium dependent. We also tested IL-1α and -1β on intracellular calcium flux using a fluorescent calcium probe. In response to both cytokines, HMVECs generated significantly greater calcium flux than PBS (Figure 5D). TTP plasma has been shown to trigger calcium-dependent endothelial degranulation.²⁹ To assess whether IL-1 present in the plasma from patients with TTP induced endothelial degranulation, we stimulated HMVECs with TTP or control plasma, in the presence or absence of anakinra. Anakinra decreased the release of VWF in the supernatants of HMVECs stimulated by TTP plasma (P = .03; Figure 5E). Consistent results were obtained from the measurement of intracellular calcium flux (Figure 5F).

Because IL-1α and -1β could trigger endothelial degranulation in vitro, we wondered whether these cytokines could induce TTP in ADAMTS13 KO mice. We injected ADAMTS13 KO and the WT control mice with high concentrations of IL-1α, IL-1β, or equivalent volume of PBS and assessed the development of TTP using previously reported readout. IL-1α and -1β resulted in significant thrombopenia compared with PBS only in ADAMTS13 KO mice (P < .001 for both; Figure 6A). To differentiate between thrombocytopenia due to microangiopathy or possible intravascular disseminated coagulation mechanism, D-dimer concentrations were measured. D-dimer increased after IL-1α and -1β injection (P < .001; supplemental Figure 6A), but this increase was independent of KO or WT genotype (P = .08), rendering unlikely an underlying intravascular disseminated coagulation mechanism in ADAMTS13 KO mice. As expected, given the short delay (10 hours), we did not observe significant anemia in the mice (supplemental Figure 6B), but schistocytes were observed in all IL-1α– and IL-1β–injected KO mice (supplemental Figure 6C). Moreover, IL-1α and IL-1β injections significantly increased VWF plasma levels only in KO mice (P < .001 for both; Figure 6B), and, as expected, this increase was associated with a loss of high-molecular-weight VWF multimers in electrophoresis (supplemental Figure 6D). IL-1α and -1β injections increased troponin levels only in KO mice (P < .001 for both; Figure 6C). Histologically, cardiac ischemic lesions were only observed in KO mice injected with IL-1α or IL-1β (P = .006 and P = .03; Figure 6D). Finally, immunofluorescence analysis of cardiac capillary microthrombi revealed that only ADAMTS13 KO mice injected with IL-1α or IL-1β exhibited significant increase in the amount of intracapillary VWF thrombi (P = .006 and P = .03, respectively; Figure 6E).

TTP is characterized by an ADAMTS13 deficiency and systemic tissue ischemia resulting from capillary microthrombus accumulation. The 2 main organs affected are the heart and brain. Cardiac ischemia is assessed in humans by blood troponin assay and echocardiographic evaluation of the ejection fraction.³⁰ It is a major risk factor for disease mortality. Assessment of neurological ischemia is not standardized to date, but recent studies have highlighted a possible alteration of the blood–brain barrier (BBB) in TTP,³¹ which may be associated with later development of cognitive impairment and depression.^32,33 

IL-1α and IL-1β are 2 main proinflammatory cytokines involved in pathogenesis of tissue lesions secondary to an ischemic process.³⁴ Indeed, the release of multiple DAMPs by ischemic dying cells induces IL-1α and -1β transcription and IL-1β processing through NLRP3 inflammasome assembly and caspase-1 activation, in large part through binding to TLR4 and TLR2.³⁵ In mice, TLR2/4 inhibition³⁶ has been shown to reduce both postischemic myocardial leukocyte infiltration and the size of myocardial infarctions.³⁷ Thus, the role of IL-1α and -1β in ischemia due to TTP deserved investigations.

First, we observed that mostly IL-1α but also IL-1β concentrations were elevated during the acute phase of the disease, normalized during remission, and correlated with disease mortality and morbidity evaluated by troponin T and PS-100b concentrations. Troponin is an important prognosis biomarker during TTP, known to correlate with cardiac involvement and mortality.³⁰ Although not previously evaluated in TTP, PS-100b is considered as a good biomarker of brain ischemia,³⁸ predicting clinical stroke status in patients during the early phase of the disease.³⁹ Circulating concentrations of PS-100b appeared to correlate with TTP-induced mortality in our study. The higher IL-1α/β ratio observed in nonsurviving patients with TTP in our study may indicate a greater contribution of IL-1α in determining the severity of TTP. IL-1α is an intracellular cytokine, constitutively present in epithelial and endothelial cells, which is released in apoptotic bodies or during cell necrosis.⁴⁰ The elevated concentrations of IL-1α observed in patients with TTP may indicate a necrotic endothelial origin of this cytokine, which is not directly related to the immune mechanism of the disease (anti-ADAMTS13 antibodies) but rather to its ischemic features. However, this study was not designed to determine the original cell source of IL-1α or -1β.

The increase in IL-1α and IL-1β levels observed in our study may seem modest. However, extremely low serum levels of these cytokines are sufficient to elicit clinically relevant proinflammatory effects (a few nanograms injected into humans are sufficient to induce fever).⁴¹ IL-1α and IL-1β exert mainly local effects (paracrine and autocrine), so serum levels are often undetectable. In a highly inflammatory disease such as severe sepsis with multiorgan failure, for example, only a few studies have succeeded in detecting a very small increase in IL-1β in the order of a few pg/mL.⁴² Similar limitations of the assays have been observed in organ ischemia such as stroke^43,44 or myocardial infarction.^45,46 So, to assess the potential pathogenic role of IL-1α and -1β during TTP, we then evaluated the effect of the recombinant human IL-1 receptor antagonist, anakinra, in a murine model of TTP. The classic murine model of TTP is based on a single injection of VWF at 2000 IU/kg.⁴⁷ This model is slightly severe (0% mortality) and failed to prove favorable effect of anakinra. We therefore adapted it by performing 3 daily injections of VWF at 1500 IU/kg, resulting in a model with high mortality much closer to that observed in humans. The dose of anakinra (100 mg/kg per day) was selected after a preliminary dose-response study. Although it was much higher than that used in humans (100 mg per day), it was consistent with doses commonly used in mouse models of ischemia^48-50 or of inflammatory diseases.⁵¹ This may be explained by a lower affinity of anakinra for the mouse IL-1 receptor. Anakinra injections decreased cardiac lesions, as assessed by troponin concentrations, echocardiography, PET imaging, and histological analysis. In addition, anakinra also reduced cerebral ischemia, demonstrated by circulating PS-100b concentrations, cerebral glucose metabolism, and BBB analysis. The cerebral and cardiac hypermetabolism ([¹⁸F]FDG uptake) observed in untreated TTP mice by PET suggests a local inflammatory response triggered by ischemia leading to vascular leakage. This was associated with a rupture of the BBB, as evidenced by capillary leakage of [^99mTc]Tc-DTPA. These results are thus in support of an important role for IL-1 in TTP pathogenesis and a protective effect of IL-1 inhibition.

Anakinra-treated mice exhibited less severe hematological disturbance than untreated mice, suggesting an unexpected role of IL-1 in endothelial VWF degranulation. We confirmed this effect in vitro using microvascular endothelial cells treated with IL-1. These findings were also observed in vivo because it was possible to induce TTP in ADAMTS13 KO mice by injection of IL-1α and -1β alone, although at supraphysiological concentrations. It should be noted that IL-1 can cause inflammatory lesions in the heart independently of ADAMTS13 deficiency. However, in this case, the histologic lesions occur only after several days,^18,52 compared with 10 hours in our study. Therefore, ADAMTS13 deficiency sensitizes cardiac cells early to IL-1 toxicity through VWF degranulation, followed by the production of microthrombi that consume platelets and high-molecular-weight VWF multimers and generate TTP.⁵³ 

Our results suggest that IL-1 may be involved in an amplification loop of VWF endothelial degranulation in TTP. Several such amplification loops have already been reported in TTP, involving complement,⁵⁴ heme,⁵⁵ or neutrophil extracellular traps⁵⁶: TTP-induced ischemia and hemolysis trigger complement activation, free heme production, and neutrophil extracellular trap generation, which promote subsequent endothelial degranulation and VWF release, amplifying formation of microthrombi and constituting a harmful amplification loop, increasing TTP severity.⁵⁷ IL-1α and -1β may also participate in such an amplification loop, because these 2 cytokines are released in response to ischemia.⁸ Thus, blocking IL-1 may not only decrease the consequences of ischemia-induced inflammation but also inhibit endothelial degranulation, a well-recognized mechanism in TTP pathogenesis⁵⁸ but not targeted by current treatments of the disease.

This study has several limitations. Cardiac and neurological outcomes in patients with TTP were assessed retrospectively and through biological markers only. In the murine model, we injected anakinra rapidly after the onset of TTP, possibly artificially increasing its effectiveness. However, given the high mortality rate in our model (80% at day 3 without treatment), it seems difficult to start IL-1 inhibition later. In addition, we did not investigate the optimal duration of anakinra treatment. Here, again, we are limited by the model because TTP is “active” only as long as VWF injections are given. As soon as these injections are stopped, surviving mice get better, and platelets rise spontaneously. It therefore appears difficult to study the effect of longer durations of anakinra treatment on remitting mice after stopping injections. To modulate the onset and duration of anakinra treatment, and possibly evaluate more time points after TTP with noninvasive Doppler and PET scans, we need to establish less severe models of microangiopathy that last several days. So far, such models are not available. Another limitation of our study is that brain [¹⁸F]FDG uptake may have been affected by BBB alteration due to TTP. Finally, the induction of TTP by IL-1 injection in mice required the administration of very high doses of IL-1. However, induction of endothelial degranulation and TTP in mice has been shown to be challenging, and only 1 such model has been reported to date, driven by large concentrations of shigatoxin.^26,59 

In conclusion, our work reports consistent results on the role of IL-1α and -1β in TTP. Released in response to ischemia, these 2 cytokines contribute to organ damage through induction of sterile inflammation and triggering VWF endothelial degranulation. Anakinra improves hematological parameters of thrombotic microangiopathy, reduces cardiac and cerebral damages, and significantly decreases TTP-induced mortality in mice. The relevance of this work should be evaluated in humans.

The authors acknowledge Cécile Denis and Eloïse Pascal for their help in von Willebrand factor (VWF) antigen and multimer analysis. The authors also thank Manal Ibrahim from the Laboratory of Hematology of Timone, Marseille, for the help in VWF and platelet reactivity.

The work was funded by INSERM, Public Assistance of the Hospital of Marseille; the National Clinical Research Project (no. 2007/23); and Société Nationale Française de Médecine Interne.

Contribution: R.M., R.C., M.L., Y.K., M.M., and E.T. conducted in vitro and in vivo experiments; R.M., M.L., S.R., and C.G. conducted the histological analyses; R.M., S.F., G.H., and B.G. performed functional imaging analyses; P.L. performed and interpreted VWF antigen and multimers analyses; P.P. and G.K. monitored the multicenter clinical study; R.M., M.L., and E.T. wrote the first draft of the manuscript; R.C., G.H., B.G., S.F., F.D.-G., and G.K. critically revised the manuscript; and R.M., E.T., and G.K. managed the overall research enterprise.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Edwige Tellier, Centre de Recherche en CardioVasculaire et Nutrition, Faculty of Pharmacy, 27 Blvd Jean Moulin, 13385 Marseille, France; email: edwige.tellier@univ-amu.fr; and Gilles Kaplanski, Internal Medicine and Immunology Department, CHU Conception, 147 Blvd Baille, 13005 Marseille, France; email: gilles.kaplanski@ap-hm.fr.