Temporal changes in macrophage phenotype after peripheral nerve injury

Abstract Background Macrophages play a key role in peripheral nerve repair and demonstrate complex phenotypes that are highly dependent on microenvironmental cues. Methods We determined temporal changes in macrophage gene expression over time using RNA sequencing after fluorescence-activated cell sorting (FACS) macrophage populations from injured peripheral nerve. We identified key upstream regulators and dominant pathways using ingenuity pathway analysis and confirmed these changes with NanoString technology. We then investigate the effects of extreme polarizers of macrophage phenotype (IL4 and IFNγ) on nerve regeneration. We determined macrophage gene expression in vivo at the site of peripheral nerve injury with NanoString technology, and assessed recovery from sciatic nerve injury by cranial tibial muscle weights and retrograde labeling motor neurons in mice with deletion of IL4 or IFNγ receptors. Results We demonstrate that IL4R and IFNγR deletions provide complementary responses to polarization, and alter expression of genes associated with angiogenesis and axonal extension, but do not influence recovery from peripheral nerve transection at 8 weeks after repair. Conclusions Overall, this study provides a framework to evaluate the phenotype of macrophages over time, and provides a broader and more precise assessment of gene expression changes than has previously been commonly used. This data suggests ways in which polarization may be modulated to improve repair

Additional file 5: of Temporal changes in macrophage phenotype after peripheral nerve injury

Figure S2. In vitro stimulation of BMDM with IFNγ+LPS or IL4 resulted in differentiation into classically activated and alternatively activated macrophage phenotypes, respectively. Cross-polarization, with each stimulus delivered sequentially, resulted in intermediate phenotypes, demonstrating macrophage plasticity. Representative dot plots of cultured BMDM from (a) an Ifngr1 −/− mouse (red) compared to a C57BL/6J mouse (black) under M(IFNγ+LPS) stimulation condition and from (b) anIl4ra −/− mouse (blue) compared to a BALB/cJ mouse (black) under M(IL4) stimulation condition with an inset histogram of the same data.Nos2 and Arg1 expression of WT strains confirm polarization to M(IFNγ+LPS) and M(IL4). (c) Median fluorescent intensity (MFI) of BMDM macrophages in the CD11b + CD16/32+ macrophage gate when exposed to 5 stimulation conditions. CD16/32 was more robustly stimulated by M(IL4) than M(IFNγ+LPS) (p < 0.0001). However, Ifngr1−/− BMDM showed the most dramatic increase in CD16/32 MFI, suggesting that IFNγ signaling may modulate the effect of LPS on CD16/32 expression. CD11b showed no clear pattern of response to stimulation, with minimal differences between strains. BALB/cJ and Il4ra −/− were analyzed separately, and Tukey groups are indicated by letters/numbers. Groups with the same letter or number are not significantly different. Significance was determined by linear mixed effect model with Tukey post-hoc comparison within stimulation. Significance was set as p < 0.05. n = 5 mice/strain. nd, no significant difference. (JPG 33 kb

Additional file 6: of Temporal changes in macrophage phenotype after peripheral nerve injury

Table S4a. Gene expression of bone marrow-derived macrophages from 3 mouse strains under 3 in vitro stimulation conditions. Log transformed mRNA transcript count mean +/−standard deviation & Tukey letters from linear mixed effect model with mouse as a random effect and an interaction term of strain*stimulation, followed by Benjamini and Hochberg false discovery rate (FDR) correction. p value for the interaction term is displayed. Table S4b. Gene expression of bone marrow-derived macrophages from 2 mouse strains under 3 in vitro stimulation conditions. Log transformed mRNA transcript count mean +/−standard deviation & Tukey letters from linear mixed effect model with mouse as a random effect and an interaction term of strain*stimulation, followed by Benjamini and Hochberg false discovery rate (FDR) correction. (ZIP 128 kb

Additional file 4: of Temporal changes in macrophage phenotype after peripheral nerve injury

Table S3. Survival after intraperitoneal endotoxin injection1. 1 Survival times of 5 mouse strains exposed to 50 mg/kg LPS I.P. Gehan–Breslow–Wilcoxon test and Mantel–Haenszel hazard ratios were calculated in GraphPad Prism. Ifngr1 −/− and IL10rb −/− were compared to their background strain C57BL/6J. Il4ra−/− were compared to their background strain, BALB/cJ. C57BL/6J were compared to BALB/cJ. (PDF 36 kb

Effect of a Histone Demethylase Inhibitor on Equine Herpesvirus-1 Activity In Vitro

Equine herpesvirus type 1 (EHV-1) is a ubiquitous and highly contagious pathogen that causes a range of disease severities with outbreaks of notable economic impact. Given the limitations in immune protection of current vaccines and the limited effectiveness of antiviral drugs on EHV-1 infections in vivo, improved treatment measures are needed to control disease. The use of drugs that alter the epigenetic state of herpes simplex virus genome has been shown to limit viral primary infection and reactivation both in vitro and in vivo. Therefore, we tested the hypothesis that maintaining a repressive epigenetic state on the EHV-1 genome in the host equine cell would decrease viral load during lytic infection. Equine fetal kidney cells (EFKCs) or isolated peripheral blood leukocytes were treated in vitro with (a) the nucleoside analog ganciclovir; (b) the histone demethylase inhibitor OG-L002; (c) both ganciclovir and OG-L002; or (d) dimethyl sulfoxide (DMSO, vehicle control); and then infected with a clinical EHV-1 isolate. Treatment of EFKCs with ganciclovir (mean 22.3 DNA copies per cell, p = 0.0005), OG-L002 (mean 25.6, p = 0.005) or both ganciclovir and OG-L002 (mean 7.1, p = 0.0001) resulted in decreased EHV-1 viral load at 24 h post-infection (hpi) in comparison with DMSO (mean 42.0), with greater impact using the combined treatment. Further, EHV-1 gene expression at 3 hpi decreased when EFKCs were infected in the presence of ganciclovir (p = 0.04) and combined treatment of ganciclovir and OG-L002 (p = 0.0003). In contrast, under similar conditions, neither ganciclovir nor OG-L002 suppressed EHV-1 infection in leukocytes. Differences between cell types, drug penetrance, or drug turnover, may have contributed to the distinct effects observed in this study