METHODS AND KITS FOR DIAGNOSIS OF FAMILIAL MEDITERRANEAN FEVER

Abstract

The present invention relates to a non-invasive, specific and rapid diagnostic method of Familial Mediterranean fever (FMF) in a subject said method comprising the step of measuring the level of cytokine (IL-18 or IL-1 beta) secreted by immune primary cells (or cell death level of these cells) obtained from said subject, which have been beforehand treated with a Protein Kinase C (PKC) inhibitor, and optionally beforehand treated with a NF-kB activator such as LPS. Inventors show based on the extensive study of the inflammasome process of the monocytes, that PKC superfamily inhibitors trigger inflammasome activation in monocytes from FMF patients while they are not sufficient to do so in monocytes from healthy donors (HD) or from patient having hyperimmunoglobulinemia D syndrome (HID S). Using cytokine release quantification or determination of real time cell death kinetics, inventors demonstrate that PKC superfamily inhibitors can discriminate FMF patients from HD or from patients with systemic inflammation from other aetiologies. These results thus set-up the basis for the development of a rapid functional specific diagnostic test for FMF. Methods of treatment are disclosed.

Claims

1. An in vitro method for diagnosing Familial Mediterranean Fever (FMF) disease in a subject, comprising the steps of i) treating immune primary cells obtained from the subject with a NF-B activator and then with a Protein Kinase C (PKC) inhibitor ii) detecting the level IL-1 beta secreted from the immune primary cells supernatant iii) comparing the level determined in step ii) with a reference value and iv) concluding that the subject suffers from Familial Mediterranean Fever when the level of IL1 beta determined at step ii) is higher than the reference value.

2. The in vitro method according to claim 1, wherein the immune primary cells are selected from the group consisting of peripheral blood mononuclear cells (PBMCs), white blood cells (WBCs), monocytes and neutrophils.

3. The in vitro method according to of claim 1, wherein the NF-B activator is Lipopolysaccharides (LPS).

4. The in vitro method according to claim 1 wherein the PKC inhibitor is selected from the group consisting of Staurosporine, UCN-01 and Ro-31-8220.

5. An in vitro method for diagnosing Familial Mediterranean Fever (FMF) disease in a subject, comprising the steps of i) treating immune primary cells obtained from the subject with a Protein Kinase C (PKC) inhibitor ii) detecting the level of IL-18 secreted from the immune primary cells iii) comparing the level determined in step ii) with a reference value and iv) concluding that the subject suffers from an Familial Mediterranean Fever when level of IL-18 determined at step ii) is higher than the reference value.

6. The in vitro method according to claim 5, wherein the immune primary cells are treated with NF-B activator prior to step i).

7. The in vitro method according to claim 6, wherein the NF-B activator is Lipopolysaccharides (LPS).

8. The in vitro method according to claim 5, wherein the immune primary cells are selected from the group consisting of peripheral blood mononuclear cells (PBMCs), white blood cells (WBCs), monocytes and neutrophils.

9. The in vitro method according to claim 5 wherein the PKC inhibitor is selected from the group consisting of Staurosporine, UCN-01 and Ro-31-8220.

10-16. (canceled)

17. Method of treating Familial Mediterranean fever (FMF) in a subject in need thereof, comprising: a) obtaining immune primary cells from the subject, b) treating the immune primary cells with a NF-B activator and then with a Protein Kinase C (PKC) superfamily inhibitor c) detecting the level of IL1 beta secreted from the immune primary cells d) comparing the level determined in step ii) with a reference value and when the IL1 beta level determined at step ii) is higher than the reference value, treating the subject with a suitable treatment.

18. The method of claim 5, wherein step of detecting is performed 3 hours or less after the step of treating.

19. The method of claim 17, wherein the suitable treatment includes one or more of administering to the subject at least one of colchicine, and IL1 antagonist, a monoclonal antibody targeting interleukin-1 beta or an interleukin 1 (IL1) receptor antagonist.

20. The method of claim 17, further comprising a step of contacting the immune primary cells with an NF-B activator prior to step b).

21. The method of claim 17, wherein the immune primary cells are selected from the group consisting of peripheral blood mononuclear cells (PBMCs), white blood cells (WBCs), monocytes and neutrophils.

22. The method of claim 20, wherein the NF-B activator is Lipopolysaccharides (LPS).

23. The method of claim 17, wherein the PKC inhibitor is selected from the group consisting of Staurosporine, UCN-01 and Ro-31-8220.

24. The method of claim 17, wherein step of detecting is performed 3 hours or less after the step of treating.

Description

FIGURES

[0090] FIG. 1: Monocytes from FMF Patients Specifically Secrete Higher Levels of IL-1 than Monocytes from Healthy Donors in Response to Various PKC Superfamily Inhibitors.

[0091] Primary monocytes (510.sup.3 per well of a 96 well plates) from HD or FMF patients presenting 2 clearly pathogenic variants (CPV) were primed with LPS (10 ng/ml) during 3 h and stimulated with (A) staurosporin (1.25 M), (B) UCN-01 (12.5 M), (C) Ro-31-8820 (100 M), (D) ATP (2.5 mM) for 1.5 h. IL-1 levels in the supernatant were determined by ELISA. Each symbol represents the average value from three technical replicates per HD (n=3 to 22 as indicated) or FMF patients (n=4 to 26 as indicated). The bar represents the mean.

[0092] FIG. 2: Monocytes from FMF Patients Die Faster than Monocytes from Healthy Donors in Response to a PKC Superfamily Inhibitor but not in Response to LPS+Nigericin Treatment.

[0093] Primary monocytes (210.sup.4 per well of a 96 well plates) from HD or FMF patients presenting 2 clearly pathogenic variants (CPV) were stimulated with (A-C) UCN-01 (12.5 M) or (D-G) LPS 10 ng/ml during 3 h followed by nigericin (5 M) in the presence of propidium iodide. Propidium iodide incorporation was monitored every 5 minutes from 5 minutes to 105 minutes post-UCN-01/nigericin stimulation using a fluorimeter. Cell death was calculated using TX-100-treated cells (100% cell death) and normalized cell death kinetics are presented (A, D). Each symbol corresponds to the average values of the normalized cell death of monocytes from 17 HD and 11 FMF patients. Mean and standard deviations are shown at each time point. (B, E, F) The Area Under the Curve (AUC) was calculated for each HD (n=17) and FMF patients (n=13) cell death kinetics from 15 to 60 min (B, E) or to 105 min (F) post-UCN-01 (B) or post-nigericin (E-F) treatment. (C-G) The time post-treatment needed to reach 20% monocytes cell death was calculated from each HD (n=17) and FMF patients (n=13) cell death kinetics. (B-C, E-G) Each symbol represents the mean value from three technical replicates per HD (n=17) or FMF patients (n=13). The bar represents the mean.

[0094] FIG. 3: Monocytes from Patients Suffering from Inflammatory Diseases Other than FMF do not Hyper-Respond PKC Superfamily Inhibitors.

[0095] Primary monocytes from HD or patients suffering from various inflammatory or infectious diseases (ID) (CAPS (n=1), HIDS (n=1), Behcet's disease (n=4), lupus (n=3), Still's disease (n=4), non-systemic juvenile idiopathic arthritis (n=1), sepsis (n=3), inflammatory bowel disease (n=4)) were (A, B) primed with LPS (10 ng/ml) during 3 h and stimulated with (A) staurosporin (1.25 M), (B) UCN-01 (12.5 M). (A-B) IL-1 levels in the supernatant were determined by ELISA. Each symbol represents the average value from three technical replicates per HD (n=16 to 22 as indicated) or ID patients (n=18). The bar represents the mean. (C-H) monocytes were stimulated with (C, E, G) UCN-01 (12.5 M) or (D, F, H) LPS 10 ng/ml during 3 h followed by nigericin (5 M) in the presence of propidium iodide. Propidium iodide incorporation was monitored every 5 minutes from 5 minutes to 105 minutes post-UCN-01/nigericin stimulation using a fluorimeter. Cell death was calculated using TX-100-treated cells (100% cell death) and normalized cell death kinetics are presented (C, D). Each symbol corresponds to the average values of the normalized cell death of monocytes from 17 HD and 18 ID patients. Mean and standard deviations are shown at each time point. (E, F) The Area Under the Curve (AUC) was calculated for each HD (n=17) and ID patients (n=18) cell death kinetics from 15 to 60 min post-UCN-01 (E) or post-nigericin (F) treatment. (G-H) The time post-treatment needed to reach 20% monocytes cell death was calculated from each HD (n=17) and ID patients (n=18) cell death kinetics. (E-H) Each symbol represents the mean value from three technical replicates per HD (n=17) or ID patients (n=18). The bar represents the mean.

[0096] FIG. 4: Monocytes from FMF Patients Presenting One Clearly Pathogenic MEFV Variant Display Heterogeneous Responses to PKC Inhibitors.

[0097] Primary monocytes from HD or FMF patients displaying a single clearly pathogenic MEFV variant (CPV) were (A) primed with LPS (10 ng/ml) during 3 h and stimulated with (A) staurosporin (1.25 M), or directly treated with (B, C) UCN-01 (12.5 M). (A) IL-1 levels in the supernatant were determined by ELISA. Each symbol represents the average value from three technical replicates per HD (n=22) or FMF patients (n=6). The bar represents the mean. The uppermost dotted line represents the average value of monocytes from FMF patients bearing two CPV. (B-C) monocytes from HD or FMF patients with the indicated genotypes were stimulated with UCN-01 (12.5 M) in the presence of propidium iodide. Propidium iodide incorporation was monitored every 5 minutes from 5 minutes to 105 minutes post-UCN-01 stimulation using a fluorimeter. Cell death was calculated using TX-100-treated cells (100% cell death) and normalized cell death kinetics are presented (B-C). Two independent experiments are shown. The average values (mean) and the standard errors of three technical replicates from one individual are shown.

[0098] FIG. 5: Monocytes from FMF Patients Die More than Monocytes from Healthy Donors in Response to a 1 h Stimulation with a PKC Superfamily Inhibitor.

[0099] Primary monocytes from HD or FMF patients were primed with LPS (10 ng/ml) during 3 h and stimulated with staurosporin (1.25 M). Cell death was determined by measuring propidium iodide incorporation/fluorescence at 1 h30 post stauroporin addition. Each symbol represents the average value from three technical replicates per HD (n=9) or FMF patients (n=7). The bar represents the mean. Cell death was normalized using TX-100-treated cells (100% cell death). With a cell death threshold set at 10% (horizontal line), this assay discriminates healthy donors from FMF patients.

[0100] FIG. 6: PKC Inhibitors-Mediated Inflammasome Activation Discriminates FMF Patients from Patients Suffering from Unrelated Inflammatory Conditions.

[0101] Receiver Operating Characteristic (ROC) curves were computed for IL-1 concentrations following (F) staurosporin or (G) UCN-01 treatment, (H) the time to obtain 20% cell death and (I) the area under the cell death kinetics curve. For each ROC curve, the AUC, the positive (ppv) and negative (npv) predictive values are indicated.

EXAMPLE

[0102] Methods:

[0103] Subjects

[0104] Twenty-six patients with FMF were included along with patients suffering from inflammatory diseases from other aetiologies (CAPS (n=1), HIDS (n=1), Behcet's disease (n=4), lupus (n=3), Still's disease (n=4), non-systemic juvenile idiopathic arthritis (n=1), sepsis (n=3), inflammatory bowel disease (n=4)) and 26 HD. All FMF patients fulfilled the Tel Hashomer criteria for FMF and had at least one mutation in the MEFV gene. The potential carriage of MEFV mutation in HD was not assessed. Blood samples from HD were drawn on the same day as patients.

[0105] Ethic Statement

[0106] The study was approved by the French Comit de Protection des Personnes (CPP, # L16-189) and by the French Comit Consultatif sur le Traitement de l'Information en matire de Recherche dans le domaine de la Sant (CCTIRS, #16.864). The authors observed a strict accordance to the Helsinki Declaration guidelines and informed written consent have been obtained from every patient or its legal representative. HD blood was provided by the Etablissement Francais du Sang in the framework of the convention #14-1820.

[0107] Monocyte Isolation

[0108] Blood was drawn in heparin-coated tubes and kept at room temperature overnight. The next day, peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Lymphocyte Separation Medium (Eurobio) [23]. Monocytes were isolated from PBMCs by magnetic selection using CD14 MicroBeads (Miltenyi Biotec) [24] and the AutoMACS Pro Separator (Miltenyi Biotec) following manufacturer's instructions. Monocytes were enumerated in the presence of a viability marker (propidium iodide, 10 g/ml) by flow cytometry (BD Accuri C6 Flow Cytometer) [25].

[0109] Inflammasome Activation

[0110] Monocytes were seeded in 96-well plates at 5103 cells/well in RPMI 1640, GlutaMAX medium (Thermofisher) supplemented with 10% fetal calf serum (Lonza). When indicated, monocytes were incubated for 3 hours in the presence of LPS (10 ng/ml, Invivogen). Unless otherwise indicated, cells were then treated for 1 h30 with nigericin (5 M, Invivogen) and ATP (2.5 mM, Sigma) [27]; staurosporin (1.25 M; Tocris); UCN-01 (12.5 M; Tocris) or Ro31-8820 (100 M; Tocris). Of note, Staurosporin from other vendors displayed 10-fold lower activity than the one we used. Following the incubation, cells were centrifuged and supernatants were collected.

[0111] Cytokine Detection and Cell Death Assay

[0112] Levels of IL-1 in monocyte supernatants were quantified by ELISA (R&D Systems). Cell death was monitored by incubating 2104 monocytes per well of a black 96 well plate (Costar, Corning) with propidium iodide (PI, Sigma) at 5 g/ml. Three technical replicates per conditions were done. UCN-01 was added at 12.5 M in the absence of any priming signal. Nigericin was added at 5 M after a 3 h priming with LPS at 10 ng/ml. Real time PI incorporation was measured every 5 minutes from 15 minutes to 105 inutes post-Nigericin/UCN-01 intoxication on a fluorimeter (Tecan) using the following wavelengths: excitation 535 nm (bandwidth 15 nm); emission 635 nm (bandwidth 15 nm) [29,30]. Cell death was normalized using PI incorporation in monocytes treated with triton X100 for 15 min (=100% cell death) and PI incorporation at each time point in untreated monocytes (0% cell death). As a further correction, the first time point of the kinetics was set to 0. The areas under the curve were computed using the trapezoid rule (Prism 6; GraphPad). To extract the time needed to reach 20% cell death, a non-linear regression analysis (Prism 6; GraphPad) was used to fit a sixth order polynomial curve to the normalized cell death kinetics using the least squares fit as the fitting method. The obtained curve was used to interpolate the time corresponding to 20% cell death.

[0113] Result

[0114] PKC Superfamily Inhibitors Trigger IL-1 Release Specifically in Monocytes from FMF Patients

[0115] We previously observed that the Pyrin inflammasome was hyper-activated in monocytes from FMF patients compared to HD in response to low doses of the bacterial toxin TcdB (Jamilloux et al. under revision). The difference in the level of IL-1 release or cell death kinetics released by monocytes from FMF patients compared to monocytes from HD was clear upon inclusion of a substantial number of FMF patients and HD. Yet, due to the inter-individual heterogeneity in the Pyrin inflammasome response, assessing the Pyrin inflammasome responses using low doses of TcdB was not sufficient to discriminate on an individual basis FMF patients from HD. We thus decided to test other Pyrin inflammasome-activating stimuli to assess whether they could allow us to discriminate functionally the monocyte response of HD and FMF patients. We first selected a cohort of FMF patients for whom the initial clinical diagnosis had been confirmed by the identification of clearly pathogenic MEFV variants (Shinar et al. 2012) on the two alleles. Patients included were either homozygous for the p.M694V or the p.M694I variants or compound heterozygous (p.[M694I];[p.V726A], p.[M694V];[p.R761H], p.[M680I];[p.V726A]). The broad specificity PKC inhibitor, staurosporin, has been reported to trigger activation of the Pyrin inflammasome in murine macrophages, likely due to the targeting of the Pyrin-inhibiting kinases PKN1/2 (Park et al. 2016). Surprisingly, in our conditions, optimized to detect signal 1+signal 2 responses (Jamilloux et al. under revision), we observed no to very low IL-1 release from monocytes isolated from HD in response to LPS (10 ng/ml) and staurosporin (1.25 M) (FIG. 1A and Fig. S1). Indeed, in our experimental conditions, monocytes from 21 out of the 22 HD released less than 50 pg/ml of IL-1 (FIG. 1A). In sharp contrast, monocytes from FMF patients released moderate to high levels of IL-1 leading to an average level 30-fold higher than the average level of healthy control monocytes (FIG. 1A). This result indicated deviating inflammasome responses to staurosporin between FMF patients and HD.

[0116] To confirm this result, we used UCN-01, a hydroxylated derivative of staurosporin, which displays a better selectivity for PKC kinases (Tamaoki 1991, 0). Similar findings were observed (FIG. 1B) with monocytes from FMF patients releasing>10-fold higher levels of IL-1 than monocytes from healthy controls. The same trends (FIG. 1C) was observed using the bisindolylmaleimide RO 31-8220, another PKC superfamily inhibitor (Davis et al. 1992). This compound was tested with only on a limited number of patients since the difference in IL-1 responses between monocytes from FMF patients and from healthy donors was lower than the one observed upon LPS+staurosporin stimulation. This lower difference might be due to the low efficacy of RO 31-8220 to inhibit PKN2 (Anastassiadis et al. 2011). As previously described (Jamilloux et al. under revision) (Van Gorp et al. 2016), we did not observe any difference in IL-1 release in response to engagement of the NLRP3 inflammasome by LPS and ATP treatment (FIG. 1D) or of the NLRC4 inflammasome (Jamilloux et al. under revision). Altogether, these results indicate that the quantification of IL-1 release in response to PKC superfamily inhibitors can discriminate monocytes from FMF patients from monocytes from HD.

[0117] Cell Death Kinetics Upon PKC Inhibitors Treatment Discriminate FMF Patients' Monocytes from HD Monocytes.

[0118] Inflammasome activation leads to the release of the proinflammatory cytokines IL-1 and triggers an inflammatory necrosis termed pyroptosis. While the release of IL-1 requires a priming step to induce the proIL-1, pyroptosis can be triggered directly upon inflammasome sensor activation. We thus assessed whether the PKC inhibitors in absence of LPS priming would trigger differential pyroptosis kinetics between monocytes from HD and monocytes from FMF patients bearing two clearly pathogenic MEFV variants. We focused on the response to UCN-01, which gave more robust results in a pilot experiment comparing various PKC inhibitors (data not shown). UCN-01 triggered a fast and strong increase in propidium iodide incorporation in the monocytes of FMF patients (FIG. 2A). In contrast, the average response was largely delayed in HD monocytes. Occasionally, we did not even notice any propidium incorporation in the monocytes of healthy donors in response to UCN-01 in the time frame of our experiment (105 min). We calculated the area under the curve (AUC) for each individual kinetics focusing on the first hour of the kinetics and the time required to reach 30% of the maximal cell death as determined using Triton X-100-mediated membrane permeabilisation. The AUC quantification demonstrated a statistical difference in the cell death kinetics of monocytes from FMF patients and healthy controls when exposed to UCN-01 (FIG. 2B). Importantly, this difference was specific for the UCN-01 inhibitor. Indeed, FMF and HD monocytes cell death kinetics overlapped perfectly upon activation of the NLRP3 inflammasome using LPS+nigericin (FIG. 2D). Furthermore, quantification of the cell death kinetics following NLRP3 engagement did not demonstrate any statistical difference in AUC between monocytes from FMF patients and healthy controls (FIG. 2E, F). AUC were calculated with kinetics stopping at either 60 minutes to be comparable with UCN-01 results or 105 minutes to better take into account the nigericin-mediated cell death kinetics. Similarly, quantification of the time needed to reach 20% cell death upon UCN-01 addition demonstrated a statistical difference between monocytes from FMF patients and healthy donors (FIG. 2C). Indeed 20% of FMF patients' monocytes died in less than 50 minutes post-UCN-01 addition while 1 h 30 was needed for monocytes from healthy controls to reach the same cell death level. In contrast, upon LPS+nigericin treatments, the level of 20% cell death was reached in 74 and 72 minutes when using monocytes from FMF patients and from healthy controls, respectively (FIG. 2F). Importantly, these results were also validated using LPS+staurosporin treatment (FIG. 5). Indeed, an average of 35% of monocytes from FMF patients were dead at 1 h 30 post-staurosporin addition while monocytes from none of the healthy donors display more than 10% cell death (set as an arbitrary diagnostic threshold-FIG. 5). These results demonstrate that monocytes from FMF patients are specifically hyper-responsive to PKC superfamily inhibitors, highlighting this functional response as of diagnostic value.

[0119] The Hyper-Response to PKC Inhibitors is Specific for Monocytes from FMF Patients.

[0120] We then assessed the responses of monocytes from patients presenting a variety of inflammatory diseases, ranging from infectious to autoinflammatory aetiologies. 20 patients were included (CAPS (n=1), HIDS (n=1), Behcet's disease (n=4), lupus (n=3), Still's disease (n=4), non-systemic juvenile idiopathic arthritis (n=1), sepsis (n=3), inflammatory bowel disease (n=4)). Monocytes from these patients were stimulated as described before. Importantly, this analysis clearly demonstrated that the hyper-responsiveness of FMF patients' monocytes to PKC inhibitors was not due to systemic inflammation but rather to a specific defect associated with FMF (FIG. 3 A-H). Of note, monocytes from CAPS patients engage the NLRP3 inflammasome in response to LPS only and reached 30% cell death before addition of the subsequent nigericin stimulus (data not shown). Yet, the kinetics of cell death of CAPS patient monocytes to UCN-01 (which is performed in absence of LPS pretreatment) were similar to the kinetics of healthy controls monocytes further strengthening the specificity of the FMF patients monocytes response to PKC inhibitors.

[0121] Monocytes from FMF patients with a single clearly pathogenic MEFV variant demonstrate a heterogeneity of functional responses to PKC inhibitors.

[0122] As previously described, genetic confirmation of the FMF clinical diagnosis is frequently impaired by the identification of a single (mono-allelic) clearly pathogenic MEFV variant. We thus decided to assess whether monocytes from patients presenting a clinical FMF diagnosis with only a single clearly pathogenic MEFV variant were hyper-responsive to PKC inhibitors. Monocytes from six FMF patients heterozygous for the p.M694V MEFV variant including one presenting on the second allele the variant of unknown significance p.E148Q were analysed for their IL-1 secretion in response to staurosporin. 4 out of the 6 patients demonstrated IL-1 levels above the 50 pg/ml threshold (FIG. 4A). The average monocyte response from patients displaying a single clearly pathogenic variant (224 pg/ml) was lower than the average monocyte response from patients displaying two clearly pathogenic variants (655 pg/ml-see the uppermost dotted line in FIG. 4A). This difference suggests a gene/variant-dosage effect consistent with the previously reported gene-dosage effects observed at the level of monocyte responses (Omenetti et al. 2014) (Jamilloux Y. et al. under revision) and clinical phenotype (Federici et al. 2012). Yet, monocytes from certain patients carrying a single clearly pathogenic MEFV variant released high IL-1 levels never observed in the supernatant of monocytes from healthy donors even when stimulated with staurosporin doses 10 times higher than the ones used in this assay (FIG. S1). This result indicates that i) the monocytes response of some patients for which genetic testing leads to ambiguous results display a clearly FMF-like signature in terms of IL-1 secretion in response to LPS+stauroporin ii) hyper-responsiveness to staurosporine is complex and not dictated only by the MEFV genotype. The hyper-responsiveness of monocytes from patient with a single clearly pathogenic variant to PKC inhibitors was confirmed using UCN-01 and the real time cell death assay in three patients bearing a single clearly pathogenic MEFV variant (FIG. 4B, 4C). Indeed, monocytes from the three FMF patients bearing a single p.M694V variant died clearly faster than monocytes from healthy controls in response to UCN-01 (FIG. 4B, 4C).

[0123] ROC Curve for IL-1 Beta

[0124] Due to the strong discrimination between FMF patients and other patients suffering from various conditions with an inflammatory component, we assessed whether the functional response to PKC inhibitors has the potential to be exploited for FMF diagnosis. We thus generated Receiver Operating Characteristic (ROC) curves to determine the sensitivity and specificity of a functional test based on IL-1 dosage following staurosporin (FIG. 6A) or UCN-01 treatment (FIG. 6B) or based on cell death kinetics parameters (time to reach 20% cell death, FIG. 6C or AUC of the cell death kinetics, FIG. 6D). The predictive values (Table 1) and the areas under the ROC curves, which are very close to 1 (1 corresponding to 100% specificity and 100% sensitivity), indicate that these functional assays accurately discriminate FMF patients from other patients presenting inflammatory conditions and from HD.

TABLE-US-00001 TABLE 1 Numerical parameters associated with the ROC curves presented in FIG. 6 Parameter Inhib. Threshold Sens. Spec. PPV NPV Acc AUC [Low-Up] IL-1 Staurosporin 44 0.85 0.88 0.88 0.84 0.86 0.93 [0.86-0.99] IL-1 UCN-01 224 0.89 0.96 0.94 0.92 0.93 0.94 [0.86-1.00] AUC.sub.RTCD UCN-01 21 0.94 1.00 1.00 0.96 0.98 0.98 [0.99-1.00] Time.sub.20% CD UCN-01 61 1.00 0.96 0.96 1.00 0.98 1.00 [0.99-1.00]

[0125] The threshold values are indicated in pg/mL-1 for IL-1 , in arbitrary units for the Area Under the real time cell death kinetics curves (AUCRTcD) and minutes for the time to reach 20% cell death (Time.sub.20% CD). Sensitivity (Sens.), Specificity (Spec.), Positive Predictive Values (PPV), Negative Predictive Values (NPV), Accuracy (Acc.) for the indicated threshold values are shown. The Area under the ROC curve (AUC) are indicated with their lower and upper values calculated using a 95% confidence interval.

[0126] Altogether, our data demonstrate that monocytes from FMF patients display enhanced inflammasome responses to PKC inhibitors paving the way to a functional diagnosis test.

Discussion

[0127] Here, we identified that monocytes from FMF patients are hyper-responsive to PKC superfamily inhibitors. This family includes PKN1/2, two kinases involved in Pyrin inflammasome signalling (Park et al. 2016). While staurosporin, UCN 01 and RO 31-8220 might target other kinases besides PKN1/2, we believe that the effects we observed here on the inflammasome are due to the targeting of PKN1/2 and the dephosphorylation of Pyrin as previously described by others (Park et al. 2016). To our knowledge, there are no inhibitors displaying a strong specificity towards PKN1/2 (Anastassiadis et al. 2011). Furthermore, genetic invalidating of PKN1/2 triggers Pyrin inflammasome activation and cell death impairing a genetic validation of the targeting of these two targets by the inhibitors tested in our assays.

[0128] Focusing on monocytes from FMF patients presenting two clearly pathogenic variants, we observed high levels of IL-1 release in response to staurosporin. Importantly, these levels were never observed in the supernatants of monocytes from healthy controls even when these monocytes were exposed to doses up to 10-fold higher of PKC inhibitors (Figure S1). This result strongly suggests that PKN1/2 inhibition is sufficient to trigger Pyrin inflammasome in monocytes from FMF patients while it is not sufficient in monocytes from healthy donors. This result is in line with a recent study by Lamkanfi and colleagues who identified that a Pyrin inflammasome regulatory step relying on microtubule dynamics is lost in PBMCs from FMF patients (Van Gorp et al. 2016). The current model for the activation of the Pyrin inflammasome in healthy donors emerging from these studies, is thus a two-step activation process requiring both dephosphorylation of Pyrin and a microtubule dynamic-dependent process. As the latter step is defective in FMF patients, dephosphorylation of Pyrin following PKC superfamily inhibitor treatment (Park et al. 2016) may explain the strong response observed in monocytes from FMF patients. The current study was performed on primary cells from patients and was thus limited to the analysis of the most frequent pathogenic variants. Future studies using genetically-engineered cell lines or a larger cohort of FMF patients with diverse genotypes are required to better understand how variations in the sequence of the Pyrin protein affect PKC inhibitors responses.

[0129] The diagnosis of FMF remains clinical and based on a number of criteria that may vary depending on ethnicity and patient age (Giancane et al. 2015). Genetic testing provides a definitive confirmation in a majority of patients through the identification of two clearly pathogenic variants (Shinar et al. 2012). Yet, there is no formal diagnosis for a large proportion of patients due to the presence of variant of unknown significance, to the presence of a single (mono-allelic) variant or even to the absence of MEFV variant. Furthermore, genetic testing is routinely a matter of weeks or months. A fast FMF diagnosis may be of particular interest as indicated by the high frequency of FMF patients with a history of appendicitis or other acute abdominal surgical interventions (Lidar et al. 2008; Samli et al. 2009). In most of these surgical operations, a typical acute abdominal attack of FMF is misdiagnosed as an acute appendicitis leading to a needless and potentially harmful procedure for the patients (Berkun et al. 2007). Furthermore, FMF is associated with a lifelong daily administration of colchicine. The question of whether colchicine can be discontinued in asymptomatic FMF patients with a single or no MEFV mutations is unresolved at the moment (Sonmez et al. 2017). Although it remains to be tested on a large cohort, a functional assay assessing the hyper-responsiveness of monocytes to PKC inhibitors might help identifying patients that may stop colchicine treatment without developing novel inflammatory symptoms.

[0130] The hyper-response to PKC inhibitors was specific of monocytes from FMF patients and was not observed in patients suffering from others autoinflammatory diseases or from microbial-mediated inflammation. Importantly, the resistance to colchicine-mediated Pyrin inflammasome inhibition was also specific of FMF patients-derived monocytes (Van Gorp et al. 2016). Two different assays based on the assessment of the Pyrin inflammasome are thus available to perform a functional FMF diagnosis. The relevance and the robustness of such a functional diagnostic test remain to be tested on a large cohort of patients with different genotypes, especially to assess the potential superiority of a functional test over the current genetic test.

REFERENCES

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