Mechanisms of action of ruxolitinib in murine models of hemophagocytic lymphohistiocytosis

Abstract

Hemophagocytic lymphohistiocytosis (HLH) is an often-fatal disorder characterized by the overactivation of T cells and macrophages that excessively produce proinflammatory cytokines, including interferon-γ (IFN-γ). Previously, we reported that the JAK inhibitor ruxolitinib dampens T-cell activation and lessens inflammation in a model of HLH in which perforin-deficient (Prf1 ^-/-) mice are infected with lymphocytic choriomeningitis virus (LCMV). Ruxolitinib inhibits signaling downstream of IFN-γ, as well as several other JAK-dependent cytokines. As a consequence, it remained unclear whether ruxolitinib was exerting its beneficial effects in HLH by inhibiting IFN-γ signaling or by targeting signaling initiated by other proinflammatory cytokines. To address this question, we compared the effects of ruxolitinib with those obtained using an IFN-γ-neutralizing antibody (αIFN-γ) in 2 murine HLH models. In both models, ruxolitinib and αIFN-γ reduced inflammation-associated anemia, indicating that ruxolitinib operates in an IFN-γ-dependent manner to reverse this HLH manifestation. In contrast, the number and activation status of T cells and neutrophils, as well as their infiltration into tissues, were significantly reduced following treatment with ruxolitinib, but they remained unchanged or were increased following treatment with αIFN-γ. Notably, despite discontinuation of ruxolitinib, LCMV-infected Prf1 ^-/- mice exhibited enhanced survival compared with mice in which αIFN-γ was discontinued. This protective effect could be mimicked by transient treatment with αIFN-γ and a neutrophil-depleting antibody. Thus, ruxolitinib operates through IFN-γ-dependent and -independent mechanisms to dampen HLH by targeting the deleterious effects of T cells and neutrophils, with the latter representing an unappreciated and understudied cell type that contributes to HLH pathogenesis.

Graphical abstract

Figure 1.

Ruxolitinib (Ruxo) targets inflammation in a murine model of secondary HLH via IFN-γ–dependent and -independent mechanisms. (A) WT C57BL/6 mice were injected with PBS (Naive) or with CpG and αIL-10R, as shown. Injected mice were left untreated (UnRx) or were treated with αIFN-γ or Ruxo on days 4 to 8 after the first CpG and αIL-10R injection. On day 9, mice were euthanized and analyzed. (B) Change in body weight (as a percentage of the initial body weight) during the course of the experiment. Body weight percentage was calculated as (actual body weight/initial body weight) × 100. Peripheral blood samples were analyzed for the number of platelets (PLT) (C) and the numbers red blood cells and for the levels of hemoglobin (Hb) (D). (E) Levels of serum cytokines were determined using Luminex. (F) Splenomegaly was assessed as a percentage of body weight and calculated as (spleen weight/actual body weight) × 100. (G) Hepatomegaly was assessed as a percentage of body weight and was calculated as: (liver weight/actual body weight) × 100. Each data point represents 1 mouse, and data were collected from 3 independent experiments. Outliers were excluded using Grubb’s test. Data shown are the mean values ± standard deviation. The total number of mice per group was n = 12 each (Naive and UnRx), n = 14 (αIFN-γ), and n = 13 (Ruxo). *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 2.

Ruxolitinib (Ruxo) targets inflammation in a murine model of primary HLH via IFN-γ–dependent and -independent mechanisms. (A) LCMV-infected Prf1^−/− mice were left untreated (UnRx) or were treated with αIFN-γ or Ruxo, as shown. On day 9, mice were euthanized and analyzed. Uninfected Prf1^−/− mice (Naive) were used as a control. (B) Change in body weight (as a percentage of initial body weight) during the course of the experiment. Body weight percentage was calculated as (actual body weight/ initial body weight) × 100. Peripheral blood samples were analyzed for the number of platelets (PLT) (C) and the number of red blood cells (RBC) and for the levels of hemoglobin (Hb) (D). (E) Levels of serum cytokines were determined using Luminex. (F) Splenomegaly was assessed as a percentage of body weight and calculated as (spleen weight/actual body weight) × 100. (G) Hepatomegaly was assessed as a percentage of overall body weight and was calculated as (liver weight/actual body weight) × 100. Each data point represents 1 mouse. Data were collected from 2 independent experiments and are shown are the mean values ± standard deviation. The total number of mice per group was n = 10 each for Naive, UnRx, αIFN-γ, and Ruxo. For cytokine analysis, the total number of mice per group was n = 6 (Naive), n = 10 (UnRx), n = 8 (αIFN-γ), and n = 9 (Ruxo). Outliers were excluded using Grubb’s test. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 3.

Ruxolitinib (Ruxo) reduces the expansion of T cells and neutrophils in secondary HLH. (A) Frequency (upper panels) and absolute numbers (lower panels) of splenic CD8⁺ T cells (left panels) and CD4⁺ T cells (right panels) gated on CD19⁻TCRβ⁺ cells. (B) Representative flow cytometric plots showing Ly6C^hiLy6G⁻ monocytes and Ly6G⁺Ly6C^int neutrophils gated on CD19⁻TCRβ⁻NK1.1⁻CD11c⁻CD11b⁺ cells. Summarized data are the frequency (upper right panels) and absolute numbers (lower right panels) of splenic monocytes and neutrophils. Each data point represents 1 mouse, and data were collected from 3 independent experiments. The mean ± standard deviation are shown. (C) Representative images showing hematoxylin and eosin–stained sections of liver (top row) and immunohistochemical-stained sections of liver showing CD3⁺ (middle row) and Neut7-4⁺ (bottom row) cells from mice injected with PBS (Naive) or CpG and αIL-10R that were left untreated (UnRx) or treated with αIFN-γ or Ruxo. Original magnification ×200. The total number of mice per group was n = 12 each (Naive and UnRx), n = 14 (IFN-γ) and n = 13 (Ruxo). (D) Summarized data from liver histological analyses showing the area of tissue infiltrated by immune cells (upper left panel), the number of inflammatory foci per field of view at 2× magnification (upper right panel) and the percentages of CD3⁺ cells (lower left panel) and Neut7-4⁺ cells (lower right panel) within inflammatory foci. Data were collected from 2 independent experiments (n = 4 mice per group). Samples were randomly chosen for histological analysis. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 4.

Ruxolitinib (Ruxo) reduces the expansion of T cells and neutrophils in primary HLH. (A) Summarized data of the frequency and absolute numbers of splenic CD8⁺ T cells (left panels), CD4⁺ T cells gated on TCRb⁺CD19⁻ live cells (middle panels), and LCMV-specific (Db gp33) CD8⁺ T cells gated on CD8⁺ T cells (right panels). (B) Frequency of splenic monocytes (Ly6C^hiLy6G⁻) and neutrophils (Ly6C^intLy6G⁺) of CD11b⁺CD11c⁻ cells. Data (mean ± standard deviation) are representative of 2 independent experiments. n = 5 mice per group. (C) Representative hematoxylin and eosin–stained liver sections (top row) and immunohistochemical staining of CD3⁺ (middle row) and Neut7-4⁺ (lower row) cells from naive Prf1^−/− mice or mice infected with LCMV that were left untreated (UnRx) or treated with αIFN-γ or Ruxo. Original magnification ×200. (D) Data were quantitated and plotted as percentage area of inflammation (upper left panel), number of inflammatory foci per field of view at 2× magnification (upper right panel) and the percentages of Neut7-4⁺ cells (lower left panel) and CD3⁺ cells (lower right panel). Data were collected from 2 independent experiments (n = 4 mice per group). Samples were randomly chosen for histological analysis. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 5.

Ruxolitinib (Ruxo) reduces CD8⁺ T-cell cytokine production in primary HLH. (A) Representative flow cytometric plots of intracellular IFN-γ and TNF-α produced by splenic CD8⁺ T cells after in vitro stimulation with LCMV-restricted gp33 peptide. Depicted on the right are summarized frequency (upper panels) and absolute numbers (lower panels) of TNF-α⁻IFN-γ⁺ and TNF-α⁺IFN-γ⁺ CD8⁺ T cells. (B) Mean fluorescence intensity (MFI) of IFN-γ in CD8⁺ T cells (left panel) and representative graphs (right panel). (C) MFI of TNF-α in CD8⁺ T cells (left panel) and representative graphs (right panel). Each data point represents 1 mouse. Data (mean ± standard deviation) are representative of 2 independent experiments (n = 5 mice per group). *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 6.

Ruxolitinib (Ruxo) reduces neutrophil activation in primary HLH. (A) Representative plots showing surface expression of TREM-1 on neutrophils. Graph depicts the frequency of splenic TREM-1⁺ neutrophils (right panel). (B) Representative plots showing intracellular TNF-α staining in splenic neutrophils gated on TCRb⁻CD11b⁺Ly6C^intLy6G⁺ cells from naive or LCMV-infected Prf1^−/− mice that were treated or not with αIFN-γ or Ruxo. Graph depicts the frequency of TNF-α⁺ neutrophils (right panel). Data (mean ± standard deviation) are representative of 2 independent experiments (n = 3 mice per group). *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 7.

Short-term treatment with ruxolitinib (Ruxo) promotes long-term survival of LCMV-infected Prf1^−/−mice. (A) Naive and LCMV-infected Prf1^−/− mice were left untreated (UnRx) or were treated with αIFN-γ on days 4 and 7 postinfection or with Ruxo on days 4 to 8 postinfection. αIFN-γ and Ruxo were discontinued on day 9, and survival was monitored until day 35. Data are combined from 3 independent experiments. P < .0001, log-rank test. (B) Mice were treated as in (A) and euthanized on day 20. For comparison, untreated LCMV-infected mice were euthanized and analyzed on day 9 (UnRx). Blood was analyzed for platelet count (PLT) (B) and for red blood cell count (RBC) and hemoglobin (Hb) (C). (D) Levels of serum cytokines. (E) Frequency (left panel) and total numbers (right panel) of splenic neutrophils on day 20 postinfection. Each data point represents 1 mouse. Data (mean ± standard deviation) are combined from 2 independent experiments. The total number of mice per group was n = 6 each (Naive and UnRx), n = 7 (αIFN-γ) and n = 8 (Ruxo). (F) Percentage survival of Naive and LCMV-infected Prf1^−/− mice left untreated (UnRx) or treated with αIFN-γ, Ruxo, neutrophil-depleting antibody (αNeut), or a combination of αNeut and αIFN-γ from days 4 to 8 postinfection, followed by treatment discontinuation. Survival was followed to day 35. Data (mean ± standard deviation) are combined from 2 independent experiments. P < .0004, log-rank test. Total number of mice examined was n = 6 each (Naive, αIFN-γ, and Ruxo), n = 3 (UnRx), and n = 9 each (αNeut and αNeut+αIFN-γ). *P < .05, **P < .01, ***P < .001, **** P < .0001.