Macrophage Heterogeneity in the Intestinal Cells of Salmon: Hints From Transcriptomic and Imaging Data

The intestine has many types of cells that are present mostly in the epithelium and lamina propria. The importance of the intestinal cells for the mammalian mucosal immune system is well-established. However, there is no in-depth information about many of the intestinal cells in teleosts. In our previous study, we reported that adherent intestinal cells (AIC) predominantly express macrophage-related genes. To gather further evidence that AIC include macrophage-like cells, we compared their phagocytic activity and morphology with those of adherent head kidney cells (AKC), previously characterized as macrophage-like cells. We also compared equally abundant as well as differentially expressed mRNAs and miRNAs between AIC and AKC. AIC had lower phagocytic activity and were larger and more circular than macrophage-like AKC. RNA-Seq data revealed that there were 18309 mRNAs, with 59 miRNAs that were equally abundant between AIC and AKC. Integrative analysis of the mRNA and miRNA transcriptomes revealed macrophage heterogeneity in both AIC and AKC. In addition, analysis of AIC and AKC transcriptomes revealed functional characteristics of mucosal and systemic macrophages. Five pairs with significant negative correlations between miRNA and mRNAs were linked to macrophages and epithelial cells and their interaction could be pointing to macrophage activation and differentiation. The potential macrophage markers suggested in this study should be investigated under different immune conditions to understand the exact macrophage phenotypes.

High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

IntroductionLung diseases are an increasing global health burden affecting millions of people worldwide. Only a few new inhaled medicines have reached the market in the last 30 years, in part due to foamy alveolar macrophage (FAM) responses observed in pre-clinical rat studies. The induction mechanism and signaling pathways involved in the development of highly vacuolated ‘foamy’ phenotype is not known. Furthermore, it has not been determined if these observations are adaptive or adverse responses.AimTo determine if high content image analysis techniques can distinguish between alveolar macrophage activation (LPS/IFN-γ activated and IL-4 activated macrophages) and if this could be applied to understanding the generation of ‘foamy’ macrophage phenotypes.MethodsNR8383 rat alveolar macrophages were stimulated with a mix of cytokines (LPS/IFN-γ or IL-4) for 24 h. The cells were further exposed to FAM inducing-compounds amiodarone and staurosporine. Following 24 h incubation, phagocytosis and lipid accumulation were measured using flow cytometry and high content image analysis techniques. The alveolar macrophages responses after exposure to cytokines were assessed by evaluation: (i) cell surface and biochemical markers such as: nitric oxide production, arginase-1 activity and MRC-1 receptor expression (ii) cellular morphology (iii) cellular functionality (phagocytic activity and lipids accumulation).ResultsMacrophages activated with LPS/IFN-γ showed distinct morphological (increased vacuolation) features and functionality (increased lipidosis, decreased phagocytic activity). Foamy macrophage phenotypes induced by amiodarone also displayed characteristics of proinflammatory macrophages (significantly increased nitric oxide production, increased vacuolation and lipidosis and decreased phagocytosis). In contrast, staurosporine treatment resulted in increased NO production, as well as arginase-1 activity.ConclusionHigh content image analysis was able to determine distinct differences in morphology between non-activated and LPS/IFN-γ activated macrophages, characterized by increased vacuolation and lipidosis. When exposed to compounds that induce a FAM phenotype, healthy non-activated macrophages displayed proinflammatory (amiodarone) or pro-apoptotic (staurosporine) characteristics but these responses were independent of a change in activation status. This technique could be applied in early drug discovery safety assessment to identify immune responses earlier and increase the understanding of alveolar macrophage responses to new molecules challenge in development of new inhalation therapies, which in turn will enhance decision-making in an early safety assessment of novel drug candidates.

Macrophage Circadian Rhythms are Differentially Affected Based on Stimuli

Macrophages are white blood cells of the innate immune system that play disparate roles in homeostasis and immune responses. As a result, they have the capability to alter their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) subtypes in response to their environment. 8-15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are also associated with cellular phenotypes, we hypothesized that the polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tested this by tracking the circadian rhythms of the mouse model macrophage cell line, RAW 264.7, which was stably transfected with Bmal1:luc and Per2:luc reporters, representing a positive and a negative component of the core molecular clock. The strength of rhythmicity was assessed using three measures: the relative power of the circadian band in the power spectral density, the rhythmicity index computed as the height of the third peak of the correlogram, and the maximum value of the chi square periodorgram. The period and amplitudes of the de-trended, smoothed time-series were estimated both by fitting to a damped cosine curve and by identifying the peak and trough of each cycle. M1 polarization decreased amplitudes and rhythmicities of both Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods and caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we then tested the circadian effects of conditioned media from two mouse breast cancer cell lines on macrophage circadian rhythms. Media from highly agressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages are known to play roles in tumors, and oncogenic features are associated with circadian rhythms, we also tested whether conditioned media from macrophages can alter circadian rhythms of cancer cells. We found that conditioned media from RAW 264.7 cells resulted in lower circadian rhythmicities and periods, but higher circadian amplitudes in human osteosarcoma, U2OS-Per2:luc cells. Taken together, our study shows that different circadian characteristics exist based on macrophage phenotypes, and suggests further that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data shows that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer cells that merits further investigation.