MS4A4A Regulates Arginase 1 Induction during Macrophage Polarization and Lung Inflammation in Mice

MS4A4A regulates the expression of arginase 1 in macrophages under IL4 stimulation. Also, MS4A4A regulates eosinophil infiltration during lung allergic inflammation induced by intranasal administration of house dust mite. Macrophages acquire the effector functions via regulating expression of a diverse array of genes [1, 2]. The allergic inflammation induces the macrophage polarization toward the cellular phenotypes of alternatively activated macrophages (AAMs) or M2 macrophages, which mediate immune responses in pathogen clearance and allergic inflammation [3, 4]. The AAMs display characteristic markers, particularly Arg1, which could be induced by IL4, or by intraperitoneal administration of chitin. MS4A4A, a member of subfamily A of the membrane-spanning, four-domain family of proteins (MS4A), has recently found to be expressed in macrophages and play important role in antitumor response [5, 6]. Interestingly, the expression of MS4A4A is upregulated by the stimulation of IL4 [5, 6]. We hypothesized that MS4A4A may regulate the functions of macrophages in the setting of allergic inflammation. To facilitate the detection of MS4A4A protein, we constructed a transgenic mouse line with an HA-tag inserted into the genomic locus of the N-terminus of Ms4a4a. mRNA of Ms4a4a was detected using quantitative PCR (qPCR) in CD11b+ cells, but not in CD3+, CD19+, or CD11b-CD3-CD19- cells isolated from the spleen and lymph nodes (Fig. 1A). Bone marrow-derived macrophages (BMDMs), peritoneal macrophages (PMs), and alveolar macrophages (AMs) all showed high levels of expression of Ms4a4a (Fig. 1A). BMDMs and PMs stimulated by IL4 showed increased expression of Ms4a4a and Arg1 (Fig. 1B, Supporting information Fig. S1A and S1B). Importantly, an immunoblot analysis with an anti-HA antibody showed that the MS4A4A protein was also upregulated (Fig. 1C and Supporting information Fig. S1C), supporting the role of MS4A4A in regulating macrophage function. To further determine the role of MS4A4A in regulating macrophage polarization and function, we used the CRISPR/Cas9 strategy to generate an Ms4a4a-deficient mouse (Supporting information Fig. S1D), and confirmed its deletion by evaluating the corresponding RNA levels in BMDMs, PMs, and other mouse tissues including lung, spleen, and peritoneal exudate cells (PECs) (Supporting information Fig. S1E and S1F). Interestingly, Ms4a4a-deficient BMDMs and PMs showed significantly lower Arg1 expression than did Ms4a4a+/+ macrophages, as indicated by both qPCR analysis (Fig. 1D and Supporting information Fig. S1G) and immunoblot analysis (Fig. 1E). Moreover, overexpression of MS4A4A in BMDMs, with MS4A4A delivered using a retroviral vector, resulted in elevated expression of Arg1 both in Ms4a4a+/- and Ms4a4a-/- macrophages, as determined using both qPCR (Fig. 1F) and immunoblot analysis (Fig. 1G). Intraperitoneal administration of chitin is adopted to elicit macrophage polarization toward AAMs and the subsequent recruitment of eosinophils in vivo [7]. The PECs were collected 48 h after the injection of chitin, and the RNA levels of related genes associated with macrophage activation and function were determined including Arg1, Ym-1, Mrc-1, Marco, and Jmjd3. The level of Ms4a4a RNA in the PECs and the total cell number of PECs were greatly increased after the chitin administration (Supporting information Fig. S2A and S2B), though no significant change was observed for the cell number of macrophages (CD11b+ F4/80+) between the heterozygous (Het, Ms4a4a+/-) and knockout (Homo, Ms4a4a-/-) groups (Supporting information Fig. S2C). The expression of Arg1 was downregulated after Ms4a4a was genetically deleted (Fig. 2A), consistent with the impaired induction of Arg1 by IL4 in culture. Of note, other relevant genes (Ym-1, Mrc-1, Marco, or Jmjd3) did not show any significant change (Supporting information Fig. S2D), which suggested a gene-specific regulatory mechanism of MS4A4A on macrophage polarization, although it remains largely unclear whether the expression of those macrophage marker genes could be regulated differentially [8]. Furthermore, the percentage of PECs made up of eosinophils (Siglec-F+CD11b+) as well as the total eosinophil number were significantly lower in the knockout mice than in the heterozygous mice, which suggested that Ms4a4a deficiency resulted in impaired eosinophil cell infiltration (Fig. 2B and Supporting information Fig. S2C). Consistent with these results, the RNA levels of eosinophil-specific genes, such as Il5ra and Ccr3, were significantly decreased in Ms4a4a-/- PECs (Supporting information Fig. S2E). Intranasal administration of HDM was then used to derive an asthma model [9] to study the role of MS4A4A for allergic inflammation. Ms4a4a, Il4, Il5, and Il13 became significantly upregulated compared to the vehicle-treated group (Fig. 2C). However, the lungs from Ms4a4a-deficient mice (Homo, Ms4a4a-/-) showed lower expression of Il4 and Il13 than did the lungs of the heterozygous (Het, Ms4a4a+/-) mice. Expression of Il5 also showed a downward trend. In addition, infiltration of eosinophils (Siglec-F+CD11c-) was decreased in the Ms4a4a-deficient mice, evidenced by their having formed a lower percentage of the total lung CD45+ immune cells analyzed using FACS (Fig. 2D and Supporting information Fig. S3A). Immunostaining of Siglec-F, which labels the populations of eosinophils and AMs, also showed decreased abundance in the lung tissues after treatment with HDMs when Ms4a4a was deleted (Fig. 2E). Thus, MS4A4A regulated the eosinophil accumulation in the allergic immune response. In summary, this study revealed the regulatory role of MS4A4A in Arg1 expression in the macrophages and allergic immunity. MS4A4A could therefore serve as a potential candidate for modulating macrophage function in various disorders such as pathogen infections and allergic inflammation. This work was supported by the National Key R&D Program of China (2017YFA0505800), the National Natural Science Foundation of China (91742106, 31822018, and 31770936), the Center for Life Sciences, and the Institute for Immunology at Tsinghua University. The authors declare no commercial or financial conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.