Anti-IL-6 cytokine treatment has no impact on elevated hematocrit or splenomegaly in a polycythemia vera mouse model

Abstract

Somatic mutations in JAK2, MPL and Calreticulin and inflammation play a key role in pathophysiology of chronic myeloproliferative neoplasia (CMN). One of the most prominent cytokines elevated in serum of Polycythemia vera patients is interleukin-6 (IL-6). Currently, it is being discussed whether suppression of inflammation by anti-cytokine approaches as anti-IL-6 treatment may be therapeutically useful in CMN. We here sought to investigate the efficacy of anti-IL-6 treatment on inflammatory cytokines, hematocrit and splenomegaly in CMN like disease. JAK2-V617F knock-in mice (JAK2+/V617F) were treated for three weeks with anti-IL-6 antibody (Ab) or IgG-control. Upon anti-IL-6 Ab treatment, serum levels of CXCL2 and CXCL10 were significantly reduced. In addition, CXCL1, CCL11, M-CSF, G-CSF, IL-17, IL-12p40 and CCL2 were reduced by a factor of 0.3 -- 0.8. Partly, this was also achieved by applying high-dose IgG. Hematocrit, erythrocyte and leukocyte counts were elevated in JAK2+/V617F mice but were not reduced by anti-IL6 Ab treatment. In addition, there was no apparent amelioration of splenomegaly and spleen histopathology. In conclusion, anti-IL-6 Ab treatment did not result in improvement of hematological disease parameters but was shown to modulate the serum cytokine signature.

Graphical abstract

Figure 1.

Analysis of serum cytokine levels and blood parameters upon anti–IL-6 Ab treatment. JAK2^+/V617F mice were treated 3 times per week over 3 weeks with 200 μg of anti-IL6 Ab per injection (MP5-20F3; BioXcell; n = 5) and IgG control (HRPN; BioXcell; n = 7). (A) The cytokine levels in serum of treated and untreated (n = 10) mice were measured by Eve Technologies Corporation (Mouse Cytokine Array/Chemokine Array 31-Plex). Cytokine level changes are shown as a heatmap according to the JAK2^+/V617F level (left) and were sorted by reduction of cytokine level by anti–IL-6 Ab treatment. Additionally, cytokines with distinct changes are shown as a scatter blot (right). (B-I) Retrobulbarly collected peripheral blood of treated and untreated mice was analyzed using the ADVIA 2120i Hematology System. Samples indicating Hct (B), RBC count (C), mean corpuscular volume (MCV) (D), platelets (PLT) (E), WBC count (F), lymphocyte (G), neutrophil granulocyte (H), and monocyte counts (I) are depicted at start and end point of anti–IL-6 Ab (n = 9) and IgG (n = 9) treatment of JAK2^+/V617F mice. *P < .05, **P < .01, nonsignificant (ns) P > .05 (by nonparametric, 2-tailed Mann-Whitney test). IFN, interferon; G-CSF, granulocyte colony-stimulating factor; M-CSF, macrophage colony-stimulating factor.

Figure 2.

Spleen analysis of untreated and treated JAK2^+/V617F mice. (A) Comparison of splenic weight measurement of untreated healthy JAK2^+/+ mice (n = 13), untreated JAK2^+/V617F mice (n = 14), and anti–IL-6– (n = 9) and IgG-treated JAK2^+/V617F mice (n = 10), respectively. Data are shown as mean ± standard error of the mean. JAK2^+/+ vs JAK2^+/V617F data are our laboratory’s internal control and are also partly reported in a manuscript by Müller et al. (B) Representative images of the isolated spleens of untreated healthy JAK2^+/+, untreated JAK2^+/V617F, and anti–IL-6 Ab– and IgG-treated JAK2^+/V617F mice are shown (scale bar, 1 cm). (C) Representative images of histopathologic spleen sections are shown (scale bar, 500 μm). Isolated spleens of untreated JAK2^+/+ and JAK2^+/V617F mice as well as anti–IL-6– and IgG-treated JAK2^+/V617F mice were stained with hematoxylin and eosin. DM6000 B Microscope (Leica Microsystems, Wetzlar, Germany) and LAS (version 3.8) software (Leica Microsystems) were used for micrographs at room temperature. ****P < .0001 by nonparametric, 2-tailed Mann-Whitney test.