Menu Close

The authors have no additional financial interests

The authors have no additional financial interests. REFERENCES 1. bone marrow chimeras were created to determine the immune-cell intrinsic role of IFNR-signaling. CD8+ T cell depletion and IL-33:ST2 blockade was performed using monoclonal antibodies. Results: LCMV contamination of mice results in severe HLH-like disease. CD8+ T cells, and the IL-33:ST2 axis remain essential mediators of disease, however, IFN-independent HLH immunopathology correlates with 10C15 fold increased neutrophilia (p 0.001) and an altered cytokine milieu dominated by IL-6, IL-1, and GM-CSF (p 0.05). Furthermore, IFN regulates CD8+ T cell expression of GM-CSF, and neutrophil survival. Conclusion: IFN is not necessary Gabapentin for development of fulminant HLH requiring physicians to consider case-by-case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL-33:ST2 signaling, may be more universally applicable treatment options that ameliorate both IFN-dependent and -impartial manifestations of HLH/MAS. INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) is usually life-threatening cytokine storm syndrome, characterized by over-abundance and over-activation of CD8+ T cells(1). The genetic abnormalities that underlie familial forms of HLH (FHL) have been traced to genes involved in the cytolytic capabilities of cytotoxic lymphocytes(2). Currently, the only effective remedy for FHL is usually bone marrow transplantation. The best characterized and most frequently identified genetic lesions occur in the perforin gene (double knockout mice with LCMV. Herein, we demonstrate that LCMV contamination is sufficient to drive HLH-like pathology in the absence of IFN. LCMV-infected double knockout mice have disease of equal lethality to Gabapentin Akt3 mice. A altered qualitative response is usually highlighted by an altered serum cytokine signature and extensive neutrophilia, reminiscent of that which is seen in Systemic Juvenile Idiopathic Arthritis (sJIA) associated MAS(17). Despite loss of IFN production, CD8+ T cells and ST2 signaling remain essential in the pathogenesis of IFN-independent HLH-like disease. Additionally, in the absence of IFN signaling, CD8+ T cells have heightened GM-CSF producing capacity, unveiling an unrecognized method of GM-CSF regulation. These results spotlight important regulatory functions for IFN in inflammatory conditions, which may be useful for diseases in which neutrophilia and/or aberrant GM-CSF production are pathogenic. Furthermore, this study addresses a clinically relevant and under-appreciated aspect of HLH biology by defining IFN-independent cytokine storm pathology. MATERIALS AND METHODS Mice C57BL/6(WT), perforin deficient(C57BL/6-Prf1tm1Sdz/J, mice, mice, and B6-CD45.1 mice. All animal studies performed with approval from The Childrens Hospital of Philadelphia Institutional Animal Care and Use Committee Induction of FHL 6 to 8-week aged mice were infected intraperitoneally with 2105 plaque-forming models of LCMV-Armstrong and euthanized upon development of significant morbidity or weight loss. Peripheral blood cell counts were performed by the Translational Core Laboratory around the Sysmex XT-2000iV Automated Hematology Analyzer at The Childrens Hospital of Philadelphia. Serum ferritin (ALPCO), soluble CD25 (R&D Systems), IL-6, GMCSF, IL-1, and IFN (BD Biosciences) were measured by ELISA. Plaque assay was performed on Vero cells as previously described(18). Histology All slides were stained with hematoxylin and eosin. Images were acquired on an Eclipse 90i microscope (Nikon, Melville, NY) using NIS-ELEMENTS software. Analysis of human gene expression Published data set (“type”:”entrez-geo”,”attrs”:”text”:”GSE26050″,”term_id”:”26050″GSE26050)(19) was accessed through the National Center for Biotechnology Information Gene Expression Omnibus. IFN-gene-set (HALLMARK_INTERFERON_GAMMA_RESPONSE) was downloaded from the MSigDB (Broad Institute). Hierarchical clustering and heatmaps were performed using Morpheus (Broad Institute). Flow cytometric analysis Peripheral blood leukocytes, splenocytes, intrahepatic leukocytes, and bone marrow leukocytes were stained with LIVE/DEAD fixable viability dye (Life Technologies), anti-CD90.2, CD19, NK1.1, CD11b, Gabapentin Ly6G, Ly6C, CD4, CD8, CD44, CD62L, CD45.2, and CD45.1 (BD Pharmingen, eBioscience, and Biolegend). H-2DbGP33C41 MHC-peptide complexes were provided as fluorophore-conjugated tetramers by Dr. E.J. Wherry (University of Pennsylvania). Samples were acquired on a MACSQuant flow cytometer (Miltenyi Biotec) and analyzed using Flowjo software version 10.4.2. Intracellular cytokine staining Splenocytes (106) were stimulated with 50ng/ml PMA (Sigma), 1g/ml ionomycin (Cell Signaling Technology), 2g/ml brefeldin A (Sigma) and 2M monensin (eBioscience) for 5 hours at 37C. After staining for LIVE/DEAD and surface antigens as before, cells were stained for IFN (clone XMG1.2 – BD) and GM-CSF (clone MP1C22E9 – BD) using the Cytofix/Cytoperm kit (BD Bioscience). In vivo antibody and recombinant IFN administration Antibodies were delivered intraperitoneally beginning day 3 post-infection and every other day thereafter. Dosing and antibodies used as following: CD8 depletion, 500g (clone 2.43, BioXcell), IgG2b isotype control (clone LTF-2, BioXcell). ST2 blockade as previously described using antibody provided by Amgen (20, 21). Cytokine blockade; 500g anti-IFN (clone XMG1.2, BioXcell), 200g anti-IL6R (clone15A7, BioXcell), 200g anti-IL-1 (clone B122, BioXcell), 100g anti-GM-CSF (clone MP1C22E9, BioXcell), and same dose of respective isotype controls (BioXcell). For recombinant-IFN give-back experiments, mice were administered 10g recombinant-IFN (Peprotech) every other day beginning either day 0, or day 6 post-infection. In vitro CD8+ T cell GM-CSF production assay Na?ve (CD44-) CD8+ T cells were enriched from na?ve mice using Na?ve CD8a+ T cell.