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Cellular and transcriptional impacts of ruxolitinib versus IFN-gamma neutralization in mouse models of HLH

ABSTRACT: Hemophagocytic lymphohistiocytosis is a cytokine storm syndrome characterized by the excessive activation of myeloid and T cells. In primary HLH (pHLH), disease pathophysiology is driven by CD8 T cells proliferation and IFNg production. Therefore, targeting IFNg has been a viable option for the treatment of HLH. Since many cytokines that signaling throught the JAK/STAT pathway, including IFNg, largely drive cytokine storm in HLH, we and others have demonstrated that targeting JAK1/2 with ruxolitinib is effective in ameliorating disease manifestations in pre-clinical models of HLH. Both IFNg and ruxolitinib treatments have also shown great efficacy in dampening inflammation in patients with HLH, albeit not completely. Since ruxolitinib treatment does not fully inhibit IFNg production, we sought to determine the effect of combining ruxolitinib treatment with IFNg neutralization as a treatment option in a LCMV-infected Prf1 mice. We aimed to elucidate the effects of these treatments on the main hematologic and cellular parameters of disease. Moreover, we determined the main changes in the transcriptional landscape on CD8 T cells isolated from mice infected with LCMV either untreated or treated with aIFNg, ruxolitinib, or combination treatments. We demonstrate that combination treatment was very effective in reversing anemia and thrombocytopenia, but failed to reduce splenomegaly, hypercytokinemia, and myeloid and T cell numbers compared to ruxolitinib treatment. Transcriptional analysis revealed distinct pathways targeted by aIFNg and ruxolitinib; while aIFNg was more effective in downregulating the expression of interferon gamma response genes, allograft rejection, and complement genes, ruxolitinib treated CD8 T cells displayed a reduction in expression of genes involved in reactive oxygen species, glycolysis, E2F targets, and protein secretion pathways. Therefore, this study provide novel insights on the effects of combining ruxolitinib treatment with IFNg neutralization in the treatment of primary HLH.

ORGANISM(S): Mus musculus

PROVIDER: GSE218500 | GEO | 2023/04/24

REPOSITORIES: GEO

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Project description:Hemophagocytic lymphohistiocytosis (HLH) comprises an expanding array of immune system disorders in which activated T and myeloid cells overproduce pro-inflammatory cytokines, many of which signal via the JAK/STAT pathway. Towards this end, use of the JAK1/2 inhibitor ruxolitinib has shown promising results in HLH pre-clinical and early phase clinical studies. Nevertheless, there remains concern for ruxolitinib-induced cytopenias, which are postulated to result from blockade of JAK2-dependent hematopoietic growth factors. To explore whether inhibition of JAK1, while sparing JAK2, might lessen inflammation and minimize hematologic toxicities in mouse models of HLH, we carried out experiments incorporating the JAK1 inhibitor itacitinib and the JAK2 inhibitor fedratinib. Itacitinib and fedratinib were well-tolerated and suppressed in vivo IFNg-mediated STAT1 phosphorylation. JAK1 inhibition with itacitinib lessened tissue infiltration of immune cells and significantly decreased serum cytokine levels of in a model of secondary (non-familial) HLH in which mice receive repeated doses of CpG DNA and IL-10 receptor blockade. However, its effects in a model of primary (familial) HLH where perforin-deficient (Prf1–/–) mice are infected with Lymphocytic Choriomeningitis virus (LCMV) were less pronounced. JAK2 inhibition with fedratinib exerted beneficial effects in both models, where it functioned similarly to ruxolitinib in improving cytopenias and reducing splenomegaly, hypercytokinemia and T cell IFNg production. RNA sequencing revealed that itacitinib induced only modest changes in gene expression in T and myeloid cells from LCMV-infected mice, while fedratinib downregulated pathways involved in cell proliferation, metabolism and oxidative phosphorylation. Neither drug rivaled the anti-inflammatory transcriptional changes seen with ruxolitinib. In summary, itacitinib and fedratinib are tolerated in mice models of primary and secondary HLH at or above clinically-relevant doses but exert differential effects with itacitinib showing partial disease improvement in the model of secondary HLH and fedratinib showing benefits similar to ruxolitinib in primary HLH.

Project description:Expression data from CD8+ T cells and CD68+ monocytes from patients with hemophagocytic lymphohistiocytosis, sepsis, and persistent systemic inflammatory response syndrome Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by pathologic immune activation in which prompt recognition and initiation of immune suppression is essential for survival. Children with HLH have many overlapping clinical features with critically ill children with sepsis and persistent systemic inflammatory response syndrome (SIRS) in whom alternative therapies are indicated. To determine if plasma biomarkers could differentiate HLH from other inflammatory conditions and to better define a ‘core inflammatory signature’ of HLH, concentrations of inflammatory plasma proteins were compared in 40 patients with HLH to 47 pediatric patients with severe sepsis or SIRS. Seventeen of 135 analytes were significantly different in HLH plasma compared to SIRS/sepsis, including increased interferon-gamma (IFNg)-regulated chemokines CXCL9, CXCL10 and CXCL11. Further, a 5-analyte plasma protein classifier including these chemokines was able to differentiate HLH from SIRS/sepsis. Gene expression in CD8+ T cells and CD68+ monocytes from blood were also enriched for IFNg pathway signatures in peripheral blood cells from patients with HLH compared to SIRS/sepsis. This study identifies differential expression of inflammatory proteins as a diagnostic strategy to identify critically ill children with HLH. Further, comprehensive unbiased analysis of inflammatory plasma proteins and global gene expression demonstrates that IFNg signaling is uniquely elevated in HLH. In addition to demonstrating the ability of diagnostic criteria for HLH, sepsis and SIRS to identify groups with distinct inflammatory patterns, results from this study support the potential for prospective evaluation of inflammatory biomarkers to aid in diagnosis of and optimizing therapeutic strategies for children with distinctive hyperinflammatory syndromes.

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Cellular and transcriptional impacts of ruxolitinib versus IFN-gamma neutralization in mouse models of HLH

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