The number of resident macrophages (MΦs) in several tissues can apparently be maintained without replenishment from blood monocytes and other hematopoietic precursors (Volkman et al., 1983; Kanitakis et al., 2004; Ajami et al., 2007; Klein et al., 2007; Murphy et al., 2008; Schulz et al., 2012; Yona et al., 2013) through in situ proliferation (Chorro et al., 2009; Davies et al., 2011; Jenkins et al., 2011; Hashimoto et al., 2013). Local proliferation restores homeostatic numbers of resident lung and peritoneal MΦs after their loss as a result of acute inflammation (Davies et al., 2011; Hashimoto et al., 2013) but can act also as an inflammatory mechanism, leading to an outgrowth of tissue-resident MΦs beyond homeostatic levels. For example, infection with the rodent filarial nematode Litomosoides sigmodontis (Ls) causes a pleuritis characterized by expansion of the resident MΦ population to high densities equivalent to that reached by recruited monocyte-derived MΦs during classical inflammation (Jenkins et al., 2011). Similarly, Langerhans cells and microglia increase in density via elevated self-renewal during atopic dermatitis and experimental autoimmune encephalitis, respectively (Chorro et al., 2009; Ajami et al., 2011). Proliferation, differentiation, and survival of MΦs are controlled by the CSF1R ligands CSF-1 and IL-34 produced by local tissue stroma to regulate the density of resident MΦs (Hume and MacDonald, 2012). CSF-1 administration to mice can increase blood monocyte and tissue MΦ numbers (Hume et al., 1988). Mice lacking the CSF1R exhibit an extreme deficit in resident MΦs in many tissues (Dai et al., 2002), and the same cells are ablated in a time-dependent manner after treatment with a blocking anti-CSF1R antibody (MacDonald et al., 2010). A proliferative signal through the CSF1R has been shown to maintain homeostatic numbers of resident peritoneal MΦs in the steady-state (Davies et al., 2013) and mediate repopulation of resident lung and peritoneal MΦs after acute inflammation or experimental depletion (Davies et al., 2013; Hashimoto et al., 2013). CSF1R signaling can also control in vivo proliferation of monocyte-derived MΦs, required for the population of the growing myometrium in pregnancy (Tagliani et al., 2011) or maintenance of recruited cells during the resolution phase of sterile peritonitis (Davies et al., 2013). The Th2 lymphokine IL-4 was first shown to regulate proliferation and accumulation of resident MΦs in the context of filarial nematode infection. Moreover, serial administration of an rIL-4 complex (IL-4c) was sufficient to induce proliferation and accumulation of MΦs throughout the body, including in the peritoneal cavity and liver (Jenkins et al., 2011), lung, and spleen (unpublished data), and to drive proliferation of recruited monocyte-derived cells (Jenkins et al., 2011). These studies did not reveal whether the actions of IL-4 were direct or indirect. Akt signaling is required for CSF-1–mediated proliferation (Smith et al., 2000; Irvine et al., 2006; Huynh et al., 2012), and we have recently shown that intact Akt signaling is critically important for in situ MΦ proliferation in response to IL-4 (Rückerl et al., 2012). However, IL-4 receptor (IL-4R) signaling does not effectively activate Akt in MΦs in vitro despite phosphorylating PKB (Heller et al., 2008), thus raising the possibility that IL-4 acts via CSF1R signaling to induce Akt-dependent MΦ proliferation. Indeed, many important parallels exist between IL-4– and CSF-1–activated MΦs. For example, both IL-4 and CSF-1 promote a suppressive and a pro-repair phenotype in MΦs (Alikhan et al., 2011). Furthermore, the transcription factors c-Myc and KLF-4 are critical for both the “alternative activation” state induced in MΦs by engagement of the IL-4R (Liao et al., 2011; Pello et al., 2012) and CSF-1–dependent proliferation that occurs in the absence of Maf B and c-Maf (Aziz et al., 2009). Thus, understanding the relationship between CSF-1 and IL-4 is important, not least because several groups have used CSF-1 to generate human “M2” MΦs (Verreck et al., 2004; Martinez et al., 2006; Fleetwood et al., 2007), which are often considered highly parallel to alternatively activated MΦs driven by IL-4. This study seeks to determine the contribution of CSF-1 to IL-4–driven MΦ proliferation and alternative activation. Alternatively activated MΦs are a distinguishing feature of inflammation driven by helminth infections and allergy but may also appear in cold-stressed adipose tissue (Nguyen et al., 2011; Karp and Murray, 2012), certain immunogenic tumors (DeNardo et al., 2009; Linde et al., 2012), and even the steady-state (Wu et al., 2011). Using direct delivery of IL-4 and Th2-biased infection models, we demonstrate that IL-4–mediated proliferation requires MΦ-intrinsic IL-4Rα signaling that is entirely independent of the CSF1R. However, these experiments revealed a significant contribution of IL-4Rα–independent, CSF1R-dependent MΦ proliferation during nematode infection. We further demonstrate that IL-4Rα expression confers a major competitive advantage to MΦs, such that IL-4Rα+ cells rapidly outcompete those lacking receptor expression. RESULTS IL-4–dependent proliferation does not require the CSF1R We used delivery of IL-4c as a reductionist approach to investigate whether IL-4 acts via the CSF1R to drive expansion of resident serous cavity MΦs. Ki67 expression was used to determine the frequency of all F4/80High MΦs in cycle, as described previously (Jenkins et al., 2011), whereas a 3-h BrdU pulse before necropsy or high level of Ki67 expression (Ki67High) was used to identify cells in S phase (Fig. S1 A; Landberg et al., 1990). Intracellular staining for RELMα and/or Ym1 was used as a marker of alternative activation. Consistent with the established role of CSF-1 in regulating steady-state MΦ levels (Davies et al., 2013), proliferation observed in control PBS-treated mice was completely blocked by treatment with anti-CSF1R mAb (Fig. 1 A). In contrast, neither elevated proliferation nor marker induction by IL-4c was affected by antibody treatment (Fig. 1 A). The only influence of blocking CSF1R on IL-4c treatment was to reduce the final MΦ number (Fig. 1 A). Daily oral gavage of GW2580, an inhibitor of the CSF1R tyrosine kinase, also had no effect on IL-4–induced MΦ proliferation or alternative activation (Fig. 1 B). Furthermore, Csf1r gene expression was significantly reduced in FACS-purified peritoneal MΦs 24 h after injection of IL-4c (Fig. 1 C). As expected, because the ligand is cleared by receptor-mediated endocytosis (Hume and MacDonald, 2012), CSF1R blockade resulted in elevated levels of CSF-1 in the tissue and bloodstream (Fig. 1 D), thereby confirming the effectiveness of the antibody. However, CSF-1 production was not increased in response to IL-4 (Fig. 1 D). Thus, in the context of IL-4 delivery, there was no evidence of CSF1R involvement in proliferation and alternative activation. Figure 1. IL-4 drives CSF-1–independent MΦ proliferation. (A) BL/6 mice were injected i.p. with IL-4c or PBS plus anti-CSF1R mAb (CSF1R), rat IgG (RIgG), or PBS on days 0 and 2. The proportion of F4/80High pleural MΦs positive for Ki67, Ki67High, or RELMα and total MΦ numbers were determined by flow cytometry on day 2 after the last injection. Graphs depict individual data for four mice/group. (B) As in A, but on day 3 after daily oral gavage with vehicle control or GW2580 on days 0–3. (C) Mice were injected with a single dose of PBS or IL-4c, and the expression of CSF1R mRNA was determined 24 h later in FACS-purified F4/80High peritoneal MΦs. ***, P 50% of the MΦs expressed IL-4Rα detectable by flow cytometry (not depicted). Infection with a GI nematode thus led to a MΦ response in the peritoneal cavity that mirrored the pleural cavity during infection with the filarial worm Ls. In both models, CSF1R and IL-4Rα contributed independently to proliferation. Figure 6. IL-4–dependent proliferation occurs during GI nematode infection and provides a competitive advantage to IL-4Rα+ MΦs. (A) BALB/c mice were infected orally with Hp, and peritoneal lavage cells were assessed at day 7. Representative flow cytograms of all peritoneal cells showing gates and frequencies for all or BrdU+, Ki67+, RELMα+ or Ym1+, F4/80High MΦs. Data are representative of five experiments. (B) BALB/c (WT) or Il4−/− (−/−) mice were infected with Hp (closed symbols) or left naive (open symbols), and the total peritoneal F4/80High MΦs and the proportion positive for BrdU, Ki67, Ym1, and RELMα+ was determined on day 7. Data are representative of three experiments with three to four mice per group. *, P 1,000-fold. Mice were injected i.p. with 20 µg Fc–CSF-1 in PBS. Where specified, mice were given 1 mg BrdU i.p. 3 h before the experimental end point. Isolation of cells from the peritoneal and pleural cavity. Mice were sacrificed by exsanguination via the brachial artery under terminal anesthesia. After sacrifice, pleural or peritoneal cavity exudate cells were obtained by washing of the cavity with lavage media comprised of RPMI 1640 containing 2 mM l-glutamine, 200 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM EDTA (Invitrogen). The first 2 or 3 ml of lavage wash supernatant from the pleural or peritoneal cavities, respectively, was frozen before analysis by ELSIA. Worm burden in the pleural lavage fluid of Ls-infected mice was determined by counting under a stereomicroscope (MZ9; Leica). Erythrocytes were removed by incubating with red blood cell lysis buffer. Cellular content of the cavities and organs was assessed by cell counting using a Casy TT cell counter (Roche) in combination with multicolor flow cytometry. Flow cytometry. Equal numbers of cells or 20 µl of blood was stained for each sample. Blood samples were mixed and washed with Hank’s buffered saline solution containing 2 mM EDTA (Invitrogen). Cells were stained with LIVE/DEAD (Invitrogen). All samples were then blocked with 5 µg/ml anti-CD16/32 (2.4G2; produced in-house) and heat-inactivated normal mouse serum (1:20) in FACS buffer (0.5% BSA and 2 mM EDTA in Dulbecco’s PBS) before surface staining on ice with antibodies to F4/80 (BM8), Siglec-F (E50-2440), Ly-6C (AL-21 or HK1.4), Gr-1 (RB6-8C5), CD11b (M1/70), CD11c (N418), MHCII (M5/114.15.2), CD19 (eBio1D3), CD4 (GK1.5), CD3 (17A2), CD115 (AFS98), IL-4Rα (mIL4R-M1), CD45.1 (A20), or CD45.2 (104; eBioscience or BD). Erythrocytes in blood samples were lysed using FACS Lyse solution (BD). Detection of intracellular RELMα, Ym1, Ki67, and BrdU was performed directly ex vivo. Cells were stained for surface markers then fixed and permeabilized using FoxP3 staining buffer set (eBioscience). For BrdU staining, cells were incubated first with or without DNase for 30 min at 37°C. Cells were then stained with biotinylated goat anti-Ym1 (R&D Systems), purified polyclonal rabbit anti-RELMα (PeproTech), or directly labeled mAbs to Ki67 (B57) or anti-BrdU (B44 or Bu20a; BD or BioLegend), followed by Zenon anti–rabbit reagent (Invitrogen) or streptavidin-conjugated fluorochromes (BioLegend). Expression of Ym1, RELMα, and Ki67 was determined relative to appropriate polyclonal or monoclonal isotype control, whereas incorporation of BrdU was determined relative to staining on non-DNase–treated cells. Analysis of proliferation and alternative activation was performed on single live cells, determined using LIVE/DEAD and forward scatter (FSC) width (FSC-W) versus FSC area (FSC-A), respectively, and subsequently gated on CD19− cells to remove B cell–MΦ doublets. Analysis for calculation of total F4/80High MΦs was performed on all cells. Samples were acquired using FACS LSRII or FACSCanto II using FACSDiva software (BD) and analyzed with FlowJo version 9 software (Tree Star). Analysis of MΦs from competitive mixed BM chimeras. MΦs derived from Cd45.1+Il4ra+/+ BM appeared to have a greater tendency to form doublets with CD19+ B cells after injection of IL-4c than cells derived from Cd45.1−Il4ra−/− BM, as judged by the high level of F4/80 expression on B cell–MΦ doublets (Fig. S2 A), a marker which is known to be up-regulated by IL-4R signaling (Jenkins et al., 2011). If doublets were excluded from analysis, such bias in doublet formation would distort the ratio of WT to Il4ra−/− MΦs. Thus, cells were treated for 5 min with Accumax cell aggregate dissociation medium (eBioscience) immediately before fixation, and population frequency analysis was performed without a single cell gate applied. To further prevent bias induced by the few remaining doublets, the gate for CD45.1+CD45.2+ cells was set to exclude events that deviated from the expected linear relationship of CD45.1 and CD45.2 on this population (Fig. S2 B, R1). Analysis of proliferation and alternative activation in these experiments was performed on CD19−F4/80High MΦs gated on single cells, using FSC-W versus FSC-A, to minimize the potential for false positives (Fig. S2 B, R2). Because of a minor bias toward more WT than Il4ra−/− pleural MΦs in naive chimeras, one-way ANOVA was used to analyze differences in cell numbers after infection, whereas paired statistical tests were used to analyze rates of proliferation and alternative activation, to better account for the variation in overall level of proliferation between mice. CSF1R experiments. Mice were injected i.p. with 0.5 mg anti-CSF1R mAb (clone AFS98), purified rat IgG control antibody, or PBS vehicle control either concurrent with IL-4c injections or on day 8 after Ls infection or day 6 after Hp infection. AFS98 mAb and purified rat IgG control were produced in-house from cultured hybridoma cells or from naive rat serum, respectively. The c-fms kinase inhibitor GW2580 (LC Laboratories; Conway et al., 2005) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer. Diluent control or 160 mg/kg GW2580 was administered daily by oral gavage from 1 h before initial dose of IL-4c on day 0 to 5 h before mice were culled on day 3. Previous pharmacokinetic analysis has shown this daily dose regimen is as effective as twice daily administration of 80 mg/kg and maintains a serum concentration of the drug over 24 h above that estimated to achieve therapeutic effect (Priceman et al., 2010). Analysis of CSF-1 in brachial arterial blood and pleural lavage fluid was performed according to the manufacturer’s instruction (PeproTech). MΦ purification and gene expression. Gene expression analysis on MΦs from IL-4c– or PBS-treated mice was performed on mRNA for which we have previously published data (Rückerl et al., 2012). In brief, 24 h after treatment of mice with PBS or IL-4c, peritoneal MΦs were sorted using a FACSAria cell sorter (BD) according to their expression of F4/80+, Siglec-F−, CD11b+, CD11c−, B220−, CD3− to purities >90%. Total RNA was then isolated using the miRNEasy kit (QIAGEN) according to the manufacturer’s instructions, measured using a NanoDrop (Thermo Fisher Scientific), and converted to cDNA with BioScript reverse transcription (Bioline) and p(dT)15 primers (Roche). Expression levels were quantified using LightCycler 480 SYBR Green I Master (Roche), with measurements performed on a LightCycler 480 (Roche). Expression of Csf1r (5′-CGAGGGAGACTCCAGCTACA-3′ and 5′-GACTGGAGAAGCCACTGTCC-3′) was normalized against Gapdh (5′-ATGACATCAAGAAGGTGGTG-3′ and 5′-CATACCAGGAAATGAGCTTG-3′). Statistics. Data were log-transformed to achieve normal distribution and equal variance where required and then tested using one-way ANOVA or Student’s t test. Where equal variance or normal distribution was not achieved, Kruskal Wallace or Spearman correlation was used. Paired Student’s t test was used to determine differences between proliferation and alternative activation of CD45.1+ and CD45.1− MΦs obtained from Ls-infected competitive mixed BM chimeric mice. Statistics were performed using Prism 5 (GraphPad Software). Each data point represents one animal. Online supplemental material. Fig. S1 shows that MΦs in S phase express the highest levels of Ki67, such that gating on Ki67High cells provides an accurate estimate of their frequency and provides representative flow cytograms of Ki67, RELMα, and Ym1 staining on peritoneal and pleural MΦs at various times after IL-4c injection. Fig. S2 shows the gating strategy used to determine frequency of MΦ subsets from competitive mixed BM chimera experiments. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20121999/DC1. Supplementary Material Supplemental Material
