CSF1R inhibition is not specific to innate immune cells but also affects T-helper cell differentiation independently of microglia depletion

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissue-resident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation. In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data.


Introduction
We recently showed that colony stimulating factor 1 receptor (CSF1R) inhibition not only affects microglia but also alters the function of bone marrow-derived macrophages [1][2][3] ; con rmed by others using CSF1R knock-out mice 4,5 and in a recent article summarizing the effects of PLX5622 in innate immune cells 6 .However, it is still unknown if these off-target effects extend cells of the adaptive immune compartment.This is critical since recent studies have employed such inhibitors to investigate interactions between innate and adaptive immune cells and have demonstrated speci c roles of T cells in CNS pathology, especially in models of autoimmune diseases and glaucoma [7][8][9] .
Here, we adopt in vivo and ex vivo models, along with targeted transcriptomics and Ingenuity Pathway Analysis (IPA), to investigate the role of CSF1R inhibition on the adaptive immune system.Here provides a brief report of the off-target effects of CSF1R inhibition, which extend to the adoptive immune system, and alert the community about the implications of these ndings in interpreting relevant experimental data.
Principal Component Analysis (PCA) of the gene array con rms that CSF1R inhibition alters the transcriptional pro le of hematopoiesis, and this change perdures long-term (Fig. 1D).Further analysis using ingenuity pathway predicts that CSF1R inhibition leads to upregulation of colony stimulating factor 2 (CSF2), which alters canonical pathways associated with Th1 and Th2 activation (Fig. 1E).
To assess if the model predictions are correct, we performed studies in CD4 + cells acquired from mice treated with PLX5622 (chow).CD4 + cells were analyzed with targeted transcriptomics to assess T helper cell differentiation pathway.Administration of CSF1R inhibitor for 3 weeks in mice profoundly affected T helper cell differentiation, as evident by transcriptional pro le changes, notably through suppression of interleukin 12 receptor subunit beta 2 (Il12rb2) and over expression of CSF2 (Fig. 2A).Further analysis using ingenuity pathway predicted that these transcriptional changes would affect Th1 and Th2 cell activation (Fig. 2B).Indeed, this prediction was con rmed by showing that CSF1R inhibition causes a dose-dependent effect on CD4 + cell survival and led to suppression of Th1/Th2 differentiation ex vivo (Fig. 2C), independently of microglia depletion.Cessation of the inhibitor restored Th1/Th2 differentiation, while IL-12 or IL-4 stimulation of CD4 + cells enhanced Th1/Th2 differentiation, respectively, in mice treated with CSF1R inhibitor (Fig. 2D-K), though mice fed with PLX5622 had lower number of CD4 + cells (Fig. 2E).

Conclusion
Here, we adopt in vivo and ex vivo models, along with targeted transcriptomics and Ingenuity Pathway Analysis (IPA), to show that CSF1R inhibition not only affects innate immune cells, especially microglia and macrophages, but also bone marrow processes that control T-cell activation and differentiation.

Materials and Methods
Data were acquired by LSRII cytometer (BD Biosciences) and analyzed by FLowJo V10 (Tree Star).
RT2 pro ler and qPCR: A total of 1 µg of RNA was used for each array analysis.cDNA was synthesized by RT2 First Strand Kit (Qiagen, Hilden, Germany) and then mixed with RT2 SYBR Green ROX qPCR Mastermix (Qiagen) before loading into the array well (Mouse hematopoiesis: PAMM-054ZA; Mouse T helper cell differentiation: PAMM-503ZA and Mouser Retinoic Acid Signaling: PAMM-180ZA).qPCR was performed by using QuantStudio 3 (ThermoFisher) according to manufacturer's manual.Data analysis was performed by using Qiagen's data analysis web portal (https://geneglobe.qiagen.com/us/analyze/) to generate a normalized expression pro le.Further normalization was performed based on 2^ (-Delta Delta CT).Gene expression heat-map, 3D PCA plot, and scatter plot were generated using the normalized gene set in R-software version 1.14.4.
Network analysis: Ingenuity Pathway Analysis (IPA, Redwood City, CA, USA) was employed to assess how gene changes effect canonical pathways, upstream regulators, and disease/functions associated 14 .A threshold of 2-fold change in gene expression and P < 0.05 was set for analysis.Data were ranked based on their z-score.
Statistical analysis: Data were analyzed with GraphPad (Prism 2.8.1, San Diego, CA) using two-tailed unpaired t-test and ordinary one-way ANOVA with Dunnet's correction for multiple comparisons.Statistical signi cance was set at P < 0.05.

Declarations
Author contributions: FL designed experiments, acquired data and analyzed data; NC, CZ analyzed data; DGV wrote and reviewed the manuscript; JC reviewed the manuscript; EIP designed experiments, analyzed data, and wrote the manuscript.
Data Availability: All data necessary for study have been included in the submission.Materials are available commercially and are listed in Materials and Methods.Although mice treated with CSF1R inhibition have lower numbers of CD4 + cells, cessation of the inhibitor ex vivo restores their ability to differentiate, though directional Th1/Th2 stimulation ex vivo enhances the response.
Mouse model: Animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the National Institutes of Health (NIH) Guidance for the Care and Use of Laboratory Animals.This study was approved by the Mass.Eye and Ear Animal Care Committee.Mice at 6-8 weeks old of both genders were used: C57BL/6J (Jackson Laboratory, Stock#: 000664).PLX5622 (PLexxikon, Inc) was formulated into AIN-76A chow (Research Diets, Inc) at the dose of 1200 ppm and given ad libitum for 3 weeks.

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