Human macrophage maturation and polarization can be monitored

The in vitro monocyte-to-macrophage maturation stood at the beginning of this thesis.

Different published maturation protocols suggested various cultivation methods and the addition of supporting cytokines, such as M-CSF [142]. However, there was reasonable concern that cultivation of monocytes in the presence of M-CSF or GM-CSF might influence the polarization capacities of mature macrophages [143-145]. It has been shown that M-CSF alone skews macrophages toward the M2 axis, and it has been hypothesized that M-CSF- induced macrophages constitute the default subtype in homeostasis in vivo [31, 146]. On the other hand, GM-CSF has been described to increase cellular responses to pro-inflammatory stimuli [147]. While M-CSF circulates at high concentrations in the blood, GM-CSF is predominant in the lung and regulates alveolar macrophage differentiation [148]. It has been described that cultivation in the presence of human serum gives rise to macrophages that resemble tissue macrophages that develop in the GM-CSF rich environment of the lung [149, 150]. In order to generate blood-derived macrophages in vitro that resemble alveolar macrophages to ensure maximal comparability with later in vivo lung studies, monocytes were cultured in the presence of human off-the-clot AB serum. Since naïve mature macrophages were of the utmost importance for unbiased polarization experiments, no auxiliary cytokine

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was given to support the monocyte-to-macrophage transition. Macrophage maturation was closely monitored by microscopy. Furthermore, the response of the cells to the polarization stimuli was examined before RNA profiling was performed. The phosphorylation of p42/44, p38, and JNK, as well as the degradation of IκBα, indicated activation of pro-inflammatory M1-associated signalling pathways, whereas phosphorylation of STAT6 pointed to M2-related cellular activation. Functional characterization of the polarization routine was furthermore achieved by infection of polarized macrophages with Legionella pneumophila and subsequent assessment of phagocytic and bactericidal capacity. As described before [151], M1 polarized macrophages showed increased uptake and killing of bacteria as compared to M0 and M2 macrophages (section 3.2).

As shown in subsequent RNA analyses, the in vitro polarization of human monocyte-derived macrophages yielded reproducible and stable miRNA and mRNA profiles that were characteristic of the respective macrophage subtype (Fig. 3.8 and 3.13). A closer investigation of mRNAs that were regulated as a response to the M1 polarization stimulus showed hallmark inflammatory gene expression such as IL1β and IL6 as well as TNFα among the most highly induced genes (Fig. 3-10). Of note, CCR7, a potential surface marker for M1 polarized macrophages that has been dismissed in cytometric screening experiments (section 3.2.3) also showed marked up-regulation on the mRNA level. This discrepancy between very high transcript occurrence and a total lack of response on extracellular protein level might either be a temporal effect, or it might be the consequence of translational repression. The 3´UTR of CCR7 mRNA was screened in silico for binding sites of highly expressed miRNAs in M1 macrophages, but no matches could be found. Finally, CD80, the surface marker eventually used for M1 macrophage identification, proofed to be strongly up-regulated on mRNA level, as expected.

The transcriptome analysis of the M2 polarized macrophages revealed CD23 as the most prominently up-regulated transcript (Fig. 3-11). The CD23 protein served as the M2 specific surface marker in this study. Furthermore, CD209 (DC-SIGN) was strongly up-regulated.

CD23 and DC-SIGN have been shown to form a cluster on chromosome 19p13 [115] and to be inducible by IL4 [116]. Up-regulated genes with a known role in alternatively activated macrophage biology furthermore included HSD11B1 and PPARγ, which have been shown to cooperate in alternative macrophage activation [117]. Recently, transglutaminase 2 (TGM2) has been found to be the only consistent and functional M2 marker upon IL4 stimulation in a comparative study of human and mouse [118].

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As these subtype-defining factors were identified in polarized macrophages that have been raised without M-CSF, it is of particular interest to note that they are in good alignment with factors found in M-CSF-generated macrophages, on condition that the same polarization stimulus is applied [112]. In the referenced publication, CD80 was identified as a M1 surface marker, while CXCL10, CXCL11, CCL5, CCR7 and IDO1 were described as M1 mRNA markers after LPS and IFNγ administration, which is in full accordance with the data gathered from the macrophages herein that were raised without M-CSF (Fig. 3-10). This analogy was less robust in M2 polarized macrophages, where PPARγ was the only shared M2 marker on mRNA level among the investigated candidates. This might be explained by the fact that in the cited study, the M2 polarization was achieved by administration of IL4 alone, whereas in our model, a combination of IL4 and IL13 was given. Hallmark IL13 inducible genes [121]

could hence be detected in the M2 polarized macrophages, such as SOCS1 and the aforementioned DC-SIGN and CD23 (FCER2), which are also described as IL4-induced.

While DC-SIGN is a typical marker of alternatively activated macrophages [152], CD23, the low affinity IgE receptor, has been implicated in allergy [52], a property that will be discussed later (section 4.4).

In summary, the obtained in vitro polarized macrophage subtypes appeared to be in good accordance with already described phenotypes.

The affirmative data on the macrophage subtype manifestation encouraged the next step, which was the miRNA profiling of the three different polarization types (M0, M1, M2). In the light of the transcriptome data (section 3.3.2), the biological variance between the samples was considered sufficiently low. However, the data gathered from the TLDA experiments (section 3.3.3) turned out to bear considerably more variation than the mRNA data, even though the same RNA samples were used. This might be due to the technical differences of the employed detection methods. The TLDA method is based on compartmentalized qPCR reactions that run in parallel in a reaction volume of 1 µl each. Gathering of the miRNA data was accomplished by performing three individual arrays per day on three different days. The time effect variance (i.e. the variance introduced by doing experiments on different days) has been described to have the most serious impact on data collection when compared to biological, between-array and within-array variation [153]. The mRNA data were collected on an Illumina HT12 Beadchip Array by probe hybridization. The mRNA samples were measured in a single run, which minimized variation due to technical inaccuracies.

Furthermore, this measurement was conducted on a standardized commercial platform (MFT

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Services, Tübingen). Additionally, while 2119 differential genes were extracted from the transcriptome data, only 43 differential miRNAs were gathered from the TLDA experiments, which further increased variance of the miRNA data due to a lower number of measurements.

Extraction of differentially regulated miRNAs and putative mRNA interaction partners therefore required extensive bioinformatic analyses. This work was done by Dr. Annalisa Marsico, assisted by Dr. Brian Caffrey, as part of a cooperation (Max Planck Institute for Molecular Genetics, Berlin).

Due to the technical challenge of extracting significantly regulated miRNAs from the array data, each potential candidate was individually validated by qPCR. As a result, hsa-miR-146a-5p, hsa-miR-146b-hsa-miR-146a-5p, hsa-miR-155-5p and hsa-miR-187-3p were shown to be up-regulated in M1 polarized macrophages, whereas hsa-miR-193b-3p and hsa-miR-511-5p were up-regulated in M2 macrophages (Fig. 3-16 and 3-17).

The evaluation of hsa-miR-34c-5p was complicated by very low or undetectable amounts in individual donor isolates. While this miRNA could be shown to be responsive to the M2 stimulus in individual samples (Fig. 3-18), the effect across all probed samples was volatile and not conclusive. In qPCR, its reliable detection required a cycling step for the pre-amplification of starting material (section 3.3.4). Hypothetically, this miRNA, if present, is subjected to down-regulation upon the M2 stimulus, but this effect apparently depends on its physiologic expression value, which seems to fluctuate between donors.