Equine alveolar macrophages and monocyte-derived macrophages respond differently to an inflammatory stimulus

Abstract

Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunophenotypes and cytokine responses to FLS stimulation is unknown. To address these questions, alveolar macrophages/monocytes (AMMs) were isolated from bronchoalveolar lavage fluid and MDMs derived from blood of six healthy horses. Separately, AMMs and MDMs were cultured with and without FLS for six hours after which cell surface marker expression and cytokine production were analyzed by flow cytometry and a bead-based multiplex assay, respectively. Results showed that regardless of exposure conditions, AMMs had significantly higher surface expression of CD163 and CD206 than MDMs. Incubation with FLS induced secretion of IL-1β, IL-8, TNF-α and IFN-γ in AMMs, and IL-8, IL-10 and TNF-α in MDMs. These results suggest that AMMs have a greater proinflammatory response to in vitro FLS stimulation than MDMs, inferring differing roles in equine lung inflammation. Variability in recruitment and function of monocyte-macrophage populations warrant more detailed in vivo investigation in both homeostatic and diseased states.

Leveraging a Comprehensive Unbiased RNAseq Database Uncovers New Human Monocyte-Derived Macrophage Subtypes Within Commonly Employed In Vitro Polarization Methods

“…However, these macrophages have largely been considered equivalent. These polarization methods have been used interchangeably and without specific thought due to the similar expression of key cell surface markers including CD80 and CD86, expression of key genes including NOS2 and PTGS2 , and secretion of inflammatory cytokines including IL6, IL8, and TNF with minimal further characterization 4,5,11,61 . Interestingly, each polarization method induced significantly increased expression of CD80 , CD86 , NOS2, PTGS2, and TNF compared to M0 macrophages; however, IFN-γ polarized macrophages consistently demonstrated the lowest fold increase in expression (data not shown).…”

“…For mechanistic in vitro studies, MDMs are commonly generated from differentiated monocytes isolated from peripheral blood [10][11][12] . Following differentiation, resulting macrophages can be polarized to M1 states using a variety of inflammatory stimuli, but are commonly generated using lipopolysaccharide (LPS), interferon gamma (IFN-γ) or a combination of both [11][12][13][14] .…”

“…For mechanistic in vitro studies, MDMs are commonly generated from differentiated monocytes isolated from peripheral blood [10][11][12] . Following differentiation, resulting macrophages can be polarized to M1 states using a variety of inflammatory stimuli, but are commonly generated using lipopolysaccharide (LPS), interferon gamma (IFN-γ) or a combination of both [11][12][13][14] . Similarly, M2 macrophages can be generated using a variety of stimuli but are commonly polarized using interleukin (IL)-4,-10,-13, or a combination of IL-4 and IL-13 [11][12][13][14] .…”

“…Following differentiation, resulting macrophages can be polarized to M1 states using a variety of inflammatory stimuli, but are commonly generated using lipopolysaccharide (LPS), interferon gamma (IFN-γ) or a combination of both [11][12][13][14] . Similarly, M2 macrophages can be generated using a variety of stimuli but are commonly polarized using interleukin (IL)-4,-10,-13, or a combination of IL-4 and IL-13 [11][12][13][14] . While the spectrum of polarization is increasingly recognized and M2 macrophages have previously been subdivided into M2a-M2d subsets depending on polarization method and subsequent function 14 , there are not universally agreed upon methods for generation of either M1 or M2 cells, and minimal investigation into potential differences in cellular responses based on which polarization method is employed in a particular study, especially for M1-type models.…”

Machine Learning Assisted Spectral Fingerprinting for Immune Cell Phenotyping

“…37 By quantifying and preventing changes in macrophage polarization states, there is potential for a decrease in disease progression. 38 To identify M1 and M2 macrophage phenotypes and quantify any potential imbalance, in both wound healing and other affected disease, flow cytometry, 37 surface marker analysis, 39 and visual morphological confirmation via optical microscopy are frequently employed. 40,41 Flow cytometry serves as a method which can rapidly differentiate different cells in a sample with highthroughput analysis.…”