Anti-Glucosylsphingosine Autoimmunity, JAK2V617F-Dependent Interleukin-1β and JAK2V617F-Independent Cytokines in Myeloproliferative Neoplasms

Abstract

Inflammatory cytokines play a major role in myeloproliferative neoplasms (MPNs) as regulators of the MPN clone and as mediators of clinical symptoms and complications. Firstly, we investigated the effect of JAK2V617F on 42 molecules linked to inflammation. For JAK2V617F-mutated patients, the JAK2V617F allele burden (%JAK2V617F) correlated with the levels of IL-1β, IL-1Rα, IP-10 and leptin in polycythemia vera (PV), and with IL-33 in ET; for all other molecules, no correlation was found. Cytokine production was also studied in the human megakaryocytic cell line UT-7. Wild-type UT-7 cells secreted 27/42 cytokines measured. UT-7 clones expressing 50% or 75% JAK2V617F were generated, in which the production of IL-1β, IP-10 and RANTES was increased; other cytokines were not affected. Secondly, we searched for causes of chronic inflammation in MPNs other than driver mutations. Since antigen-driven selection is increasingly implicated in the pathogenesis of blood malignancies, we investigated whether proinflammatory glucosylsphingosine (GlcSph) may play a role in MPNs. We report that 20% (15/75) of MPN patients presented with anti-GlcSph IgGs, distinguished by elevated levels of 11 cytokines. In summary, only IL-1β and IP-10 were linked to JAK2V617F both in patients and in UT-7 cells; other inflammation-linked cytokines in excess in MPNs were not. For subsets of MPN patients, a possible cause of inflammation may be auto-immunity against glucolipids.

Keywords: CALR exon 9 mutants; CRISPR technology; IL-1Rα; IL-33; IP-10; JAK2V617F; UT-7; antigenic stimulation; auto-immunity; cytokines; glucolipids; glucosylsphingosine (GlcSph); inflammation; interleukin-1β (IL-1β); leptin; myeloproliferative neoplasms (MPNs).

Figure 1

Differences in cytokine levels between PV, ET and primary myelofibrosis (PMF). Significant differences were found between PV, ET or PMF patients in the levels of 12 cytokines: (a) IL-1Rα; (b) IL-4; (c) IL-9; (d) IL-17; (e) IL-26; (f) MIG; (g) TGF-β₁; (h) TGF-β₂; (i) TGF-β₃; (j) HGF; (k) IL-15; (l) IL-6. Results are presented as the means + SEM. NS: not significant. (*) p < 0.05 and (**) p < 0.01, Mann–Whitney t-test. Dotted blue lines represent the upper normal values for healthy individuals according to the manufacturers of the BioPlex Pro-human Cytokine kits, measured in 66 healthy donors. Dotted green lines represent the upper normal values for healthy individuals measured in the 17 healthy donors of our control cohort.

Figure 2

Correlations between cytokine levels and % of JAK2V617F-mutated alleles. Analysis of the cytokine levels and %JAK2V617F of the 55 patients with JAK2V617F-mutated MPN, using Spearman’s t-test, revealed positive correlations between %JAK2V617F and IP-10 (a) and IL-1Rα (b), and a negative correlation between leptin and %JAK2V617F, in PV only (c). (d–f) Positive correlations in PV only between the %JAK2V617F and IP-10 (d), IL-1Rα (e) and IL-1β (f). (g) Positive correlation between IL-33 and %JAK2V617F in ET only.

Figure 3

Differences in cytokine levels in CALR- and JAK2V617F-mutated ET. Significant differences were found between CALR- and JAK2V617F- mutated ET for 9 cytokines: (a) IL-9; (b) IL-4; (c) IL-26; (d) TGF-β₂; (e) TGF-β₃; (f) IL-1Rα; (g) IL-1β; (h) TNF-α; (i) IFN-α₂; (j) b-FGF; (k) IL-5. Results are presented as the means + SEM; note the changes in scales (Y axis). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 and (****) p < 0.0001, Mann–Whitney t-test. Dotted blue lines represent the upper normal values for healthy individuals according to the manufacturers of the BioPlex Pro-human Cytokine kits, measured in 66 healthy donors. Dotted green lines represent the upper normal values for healthy individuals as measured in our control cohort of 17 healthy donors.

Figure 4

Cytokine production of wild-type JAK2 and JAK2V617F UT-7 cells. (a) The basal level of 40 cytokines and chemokines and two soluble receptors were quantified in the supernatants of wild-type JAK2 UT-7 cells; note the difference in scale for nine cytokines, produced at high levels (left panel). Results are presented as the means + SEM. (b) Only three molecules out of 42 measured in quadriplates in cell supernatants were found to be significantly more secreted in the supernatants of UT-7 clones expressing at least 50% JAK2V617F, compared to wild-type JAK2 UT-7 cells; note the difference in scale for IL-1β, produced at very low levels by UT-7 cells (left panel). Median values are represented by black bars. (*) p < 0.05, Mann–Whitney t-test. In these experiments, UT-7 cells were grown in the presence of GM-CSF.

Figure 5

GlcSph-reactivity of serum IgGs from PV, ET, PMF patients. GlcSph-specific immunoblotting assays were performed as described in Methods. (a,b,d) Both the gel of serum protein electrophoresis (SPE), after coloration, and the result of the GlcSph immunoblot, revealed by chemiluminescence, are shown; note that migration pattern may vary. (a) Example of 2/54 HDs with no GlcSph-reactive IgG in serum. (b) Examples of 5/60 MPN patients with no GlcSph-reactive IgG in serum. (c) 15/15 MPN patients with GlcSph-reactive IgGs in serum.

Figure 6

Cytokine levels according to the presence of GlcSph-reactive IgGs. Significant differences in the serum levels of 11 cytokines were found between MPN patients with GlcSph-reactive IgGs (+) and MPN patients with no GlcSph-reactive IgGs (-), as indicated. Results are presented as the means + SEM. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 and (****) p < 0.0001, Mann–Whitney t-test. Dotted blue lines represent the upper normal values observed for healthy individuals according to the manufacturers of the BioPlex Pro-human Cytokine kits, measured in 66 healthy donors. Dotted green lines represent the upper normal values for healthy individuals as measured in our control cohort of 17 healthy donors.

Figure 7

GlcSph levels measured in the blood serum of MPN patients. GlcSph−: MPN patients without GlcSph-reactive IgGs; GlcSph+: MPN patients with GlcSph-reactive IgGs. (*) p < 0.05, (***) p < 0.001, Mann–Whitney t-test. Median values are represented by black bars. The dotted blue line represents the upper normal value for healthy individuals (1.8 nmol/L). The dotted green line represents the upper value of our control cohort of HDs.

Figure 8

JAK2V617F-dependent vs. mutation-independent inflammation in MPNs. At least 26 cytokines are overproduced in MPNs. CALR mutants did not alter cytokine production, whereas JAKV617F increased the expression of IL-1Rα, IL-1β and IP-10. Other cytokines overexpressed in MPNs are presumably produced by nonmutated cells, and this type of inflammation is likely to precede the acquisition of JAK2/STAT5 activating mutations. An autoimmune response to GlcSph, observed in 20% of MPNs, could be an early cause of chronic inflammation—and excessive IL-1β production—eventually leading to the acquisition of JAK2 or CALR mutations and MPN development. In JAK2V617F-mutated MPNs, strongly inflammatory IL-1β associated with IL-1Rα expression, both induced by JAK2V617F, further stimulate cytokine production by both nonmutated cells of the bone marrow environment and mutated cells. JAK2V617F-mutated cells also secrete TNF-α, known to enhance clonal expansion. Neither IL-1β nor TNF-α signal via JAK/STAT, and thus are not sensitive to JAK inhibitors. In contrast, IFN-α represses IL-1β, and also HGF, an autocrine survival factor for clonal MPN progenitors. In addition, IFN-α stimulates T-lymphocyte subsets that secrete anti-inflammatory IL-4, and two prothrombopoiesis and prothrombosis cytokines—IL-9 (an inducer of IL-4) and IL-17.