Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo¹

Mice, FL transplantation, competitive repopulation, and peritoneal macrophages

Mouse BM cells were harvested from 6 to 10 wk old C57BL/6 (CD45.2⁺) and C57BL/6-Ly5.2 (CD45.1⁺) mice by flushing femurs and tibias. FL cells were isolated from embryonic (postcoital) day (E) 14.5 KLF4^−/− (19) and wt littermates (on a C57BL/6 background, provided by Dr. Julie Segre, National Institutes of Health, Bethesda, MD) and genotyped by PCR. To generate hematopoietic chimeras, 1–2 × 10⁶ KLF4^−/− or wt FL cells (CD45.2⁺) were transplanted i.v. into 6–8-wk-old lethally (1000 cGy) irradiated CD45.1⁺ hosts. For competitive repopulation experiments, 5 × 10⁵ FL cells (CD45.2⁺) plus 5 × 10⁵ wt BM cells (CD45.1⁺) were co-transplanted into lethally irradiated CD45.1⁺ hosts.

Peritoneal macrophages were isolated 8–12 wk after FL transplant by flushing the peritoneal cavity with 10 ml PBS. For inflammation experiments, 0.75 ml 4% brewer’s thioglycolate (Sigma-Aldrich) was injected into the peritoneum 3 days before peritoneal macrophage harvest. Three × 10⁵ macrophages were plated in each well of a 96-well flat-bottom plate and allowed to adhere overnight. Then, wells were washed three times with fresh medium to remove nonadherent cells. All studies performed in mice were approved by the Johns Hopkins University Animal Care and Use Committee.

Cell lines and culture

HL60 (American Type Culture Collection) human leukemia cells and primary mouse peritoneal macrophages were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS (FBS; Gemini Bio-Products), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen Life Technologies). Stable cell lines transduced to express a KLF4-estrogen receptor (ER) fusion and GFP (see below) were enriched by FACS-sorting for GFP⁺ cells, followed by limiting dilution cloning. At least three clones were examined for each construct. In brief, 4-hydroxy-tamoxifen (4HT; in 100% ethanol) was added to cell cultures to achieve a final concentration of 200 nM. TPA and all-trans-RA were purchased from Sigma-Aldrich and resuspended in DMSO or ethanol, respectively. For hematopoietic colony-forming cell (CFC) assays, 5 × 10⁴ mouse FL or BM cells were plated in methylcellulose medium (Stem Cell Technologies) supplemented with 50 ng/ml recombinant mouse stem cell factor (SCF), 10 ng/ml recombinant mouse IL-3, 10 ng/ml recombinant human IL-6, and 3 U/ml recombinant human erythropoietin (all cytokines from PeproTech). Monocyte-macrophage-specific CFC assays were performed as above, except medium was supplemented only with 50 ng/ml SCF and 10 ng/ml recombinant mouse monocyte-CSF (M-CSF). Colonies were counted 7 days after plating.

Plasmid constructs and dual promoter lentiviral vectors

The human KLF4 open reading frame was generated by RT-PCR, and cloned into an intermediate vector downstream of an elongation factor 1α promoter. A modified ligand-binding domain construct of the mouse ER, which is selectively responsive to 4HT (28), was fused to the 3′ terminus of KLF4 (KLF4-ER). The elongation factor 1α α-KLF4-ER cassette was then subcloned into the FUGW (29) lentiviral plasmid (provided by Dr. David Baltimore, California Institute of Technology, Pasadena, CA). All clones were sequence verified. Lentiviral packaging and transductions were conducted as described previously (30, 31).

Flow cytometric analysis and cell purification

The following FITC-, PE-, PerCP-Cy5.5-, or allophycocyanin-coupled mAbs against mouse leukocyte differentiation Ags were obtained from BD Biosciences: CD3, CD4, CD8, CD45R/B220, CD45.1, CD45.2, CD69, NK1.1, and Gr1 (recognizes Ly6C and Ly6G). CD115 and F4/80 mAbs were purchased from eBioscience. Blood samples were lysed with RBC lysis buffer (eBioscience), and then cells were blocked with Fc Block (BD Biosciences), stained at 4°C with optimal amounts of fluorochrome-coupled mAbs diluted in staining medium (PBS containing 2.5% FBS) for 30 min, then washed. At least 20,000 events were acquired per immunostain on a FACSort or FACSCalibur (BD Biosciences) flow cytometer. Similarly, levels of competitive repopulation were measured using CD45.1-PE and CD45.2-FITC mAbs (BD Biosciences). Percent engraftment of donor CD45.2⁺ FL cells was calculated as: 100 × (number of CD45.2⁺ cells)/(number of CD45.1⁺ cells + number of CD45.2⁺ cells). For cell fractionation, whole BM or splenocyte suspensions were RBC lysed and stained with mAbs specific for T cells, B cells, NK cells, erythroid progenitors, and monocytes, then enriched by cell sorting on a FACSVantage or FACSAria (BD Biosciences) flow cytometer. CD115⁺ cells were enriched, for Western blotting, using anti-biotin MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol. Enrichment by positive selection was verified (>40% CD115⁺) by flow cytometry.

Control or genetically modified HL60 cells were stained using propidium iodide (PI; Sigma-Aldrich) at selected time points after addition of 4HT or vehicle control (32). DNA content analysis was performed using the Dean-Jett-Fox model in the FlowJo FACS analysis software (Tree Star).

Macrophage activation and Griess assay

Adherent peritoneal macrophages (described above) were treated with various concentrations of LPS (Sigma-Aldrich) at 37°C for 24 h. The generation of nitrite was used as a measure of NO release from peritoneal macrophages; nitrite concentrations in supernatants were measured by the Griess reaction as described by the manufacturer’s protocol (Promega). RNA was harvested from cells 24 h after treatment with LPS.

qRT-PCR amplification and Western blots

RNA was isolated using the RNAeasy kit (Qiagen), and Superscript III (Invitrogen Life Technologies) was used to make cDNA, according to the manufacturer’s protocols. Real-time quantitative-RT-PCR (qRT-PCR) was conducted as described previously (27). Unless stated otherwise, qRT-PCRs were normalized with hydroxymethylbilane synthase expression. Sequences for all primers used for qRT-PCR are available upon request.

Western blots were performed using standard procedures with the following modifications. Cells were cultured in complete medium supplemented with MG132 (30 μM; Sigma-Aldrich) for 5 h before harvesting (33). Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with complete mini tablets (Roche) and lysates were quantified and loaded immediately. Polyclonal Abs recognizing KLF4 and GAPDH were purchased from Santa Cruz Biotechnology and Abcam, respectively. Densitometry was performed on a Bio-Rad GS-800 Calibrated Densitometer using Quantity One program from Bio-Rad.

KLF4 was expressed in HSCs and monocyte-macrophages

We previously reported KLF4 among the transcripts over-expressed in human HSC-enriched populations (CD34⁺/[CD38/Lin]^low), as compared with HPC-enriched populations (CD34⁺/[CD38/Lin]^high) (27), and similar results have been found in mice (34). To further characterize the hematopoietic expression of KLF4 in mouse cells, we isolated several types of committed blood and immune cells from wt C57BL/6 mice, and we measured the expression of KLF4, and for comparison, its two closest family members, KLF1 and KLF2 (Fig. 1, A–C). As anticipated (35), KLF1 was highly expressed only in mouse BM erythroid cells. KLF2 was expressed in all mouse lymphoid populations, but at low levels in monocytic and erythroid BM cells. KLF4 was expressed most highly in mouse BM monocytic cells (CD45⁺CD115⁺Gr1⁺), consistent with previous microarray experiments in humans, which found the highest levels of KLF4 transcripts in CD14⁺ monocytes (36). We found intermediate levels of KLF4 in NK cells and B cells and low levels in T cells and erythroid BM cells. We confirmed that KLF4 protein was expressed in cell lysates from whole BM, spleen, thymus, and CD115⁺ purified cells (Fig. 1, E and F). We next measured the expression of KLF4 in activated peritoneal macrophages. As has been demonstrated in human cells (17), activation of mouse macrophages using LPS induced KLF4 expression (Fig. 1D).

KLF4^−/− FLs contained normal numbers of functional HPCs and HSCs

KLF4^−/− mice die shortly after birth due to an epithelial defect (19), but have been reported to have blood. We found that FLs from E14.5 KLF4^−/− embryos had similar numbers of total cells as wt embryos (data not shown). FL cells from KLF4^−/− embryos formed similar numbers of in vitro granulocyte/monocyte (CFC-GM) and granulocyte/monocyte/erythroid (CFC-Mix) colonies, and slightly more erythroid (BFU-E) colonies (Fig. 2A). To examine the ability of FL cells from wt and KLF4^−/− embryos to form monocytic/macrophage (CFC-M) colonies specifically, we cultured FL cells in methylcellulose containing only M-CSF and SCF; there were no significant differences in the numbers of CFC-M from wt or KLF4^−/− embryos (data not shown). In several experiments, we observed only slight differences between KLF4^+/+ and KLF4^+/− mice, so we have grouped these in certain (indicated) experiments, such as Fig. 2A.

To examine the in vivo hematopoietic capacity of KLF4^−/− cells, we performed transplants of wt or KLF4^−/− FL cells into lethally irradiated congenic adult recipient mice. KLF4^−/− cells provided radioprotection, and complete blood cell counts performed 6 and 10 wk after transplant showed no significant differences between mice transplanted with wt or KLF4^−/− FL cells (Fig. 2C). To assess competitive engraftment and repopulation ability, equal numbers of wt or KLF4^−/− FL donor cells (CD45.2⁺) were cotransplanted with congenic (CD45.1⁺) wt BM donor cells into lethally irradiated congenic (CD45.1⁺) adult recipient mice, and FL cell-derived (CD45.2⁺) engraftment levels were measured over a 40 wk time course. We observed no significant differences in engraftment/repopulation by KLF4^−/− compared with wt FL cells (Fig. 2B).

KLF4 was essential for normal late monocytic differentiation in vivo

Because we found high levels of KLF4 in BM monocytic cells, we next examined the BM, PB, spleen, and peritoneal fluid of KLF4^{+/+ or +/−} and KLF4^−/− chimeras for monocyte-macrophages (Fig. 3). KLF4^−/− chimeras had a 43% reduction in the number of donor-derived (CD45.2⁺) BM monocytic cells. Peripheral blood from KLF4^−/− chimeras had a 53% reduction in the number of donor-derived resident monocytes (CD115⁺Gr1⁻) and was essentially void of donor-derived inflammatory monocytes (CD115⁺Gr1⁺; Fig. 3A middle right panel with arrow). Spleens from KLF4^−/− chimeras had essentially no donor-derived inflammatory monocytes, but the numbers of donor-derived resident monocytes were unaffected. The numbers of donor-derived peritoneal macrophages were unaffected by loss of KLF4. Examination of other cell lineages showed no significant differences.

We next sought to understand the cause of the decreased numbers of monocytic cells in the KLF4^−/− chimeras. Because KLF4^−/− FL cells showed no defect in colony formation in vitro, we examined donor-derived monocytic cells from KLF4^−/− chimeras to see whether loss of KLF4 affected the survival of these cells in vivo. Although there were no differences in the numbers of Annexin V-positive granulocytic or lymphoid cells, there was a 2-fold increase in the numbers of Annexin V-positive monocytic cells in the BMs of KLF4^−/− chimeras, suggesting that KLF4 may be involved in the survival of monocytic progenitors.

Loss of KLF4 disrupted expression of key trafficking molecules

Recent reports demonstrated that the closely related KLF2 transcription factor regulates several critical trafficking molecules in T lymphocytes (3, 4). Therefore, we measured the expression of several molecules (previously shown to be regulated by KLF2) in KLF4^−/− monocytic cells. Donor-derived BM monocytic cells from KLF4^−/− chimeras expressed lower levels of β₇ integrin and the inflammatory monocyte marker CD62L, but expressed slightly higher levels of F4/80, a late monocyte-macrophage differentiation marker (Fig. 4A); there was no effect on the level of CD69. Consistent with our flow cytometric measurements, BM monocytic cells from KLF4^−/− chimeras had lower levels of β₇ integrin and CD62L mRNA by qRT-PCR (Fig. 4C); levels of CCR2 were reduced 6-fold in the in KLF4^−/− monocytic cells.

KLF4^−/− macrophages became activated in response to LPS

Previous reports have shown that KLF4 is an important mediator of proinflammatory signaling in human macrophages in vitro (17). Thioglycolate-induced peritoneal macrophages from KLF4^+/+ and KLF4^−/− chimeras became larger and contained cytoplasmic granules after culture in the presence of LPS, indicating similar activation. In addition, we found no significant reduction between KLF4^+/+ and KLF4^−/− macrophages in nitrite production (Fig. 5A) or TNF-α mRNA up-regulation.

Induction of KLF4 expression in HL60 cells stimulated monocytic differentiation, as well as p21-associated proliferative arrest

To determine whether KLF4 is sufficient to regulate components of monocyte-macrophage differentiation, we examined the effects of enforced KLF4 expression on the HL60 human leukemia cell line, as a model of granulocytic or monocyte-macrophage differentiation. We were unable to obtain an HL60 cell line which stably expressed KLF4, suggesting that constitutive KLF4 expression was lethal or growth inhibitory. Therefore, we used an approach that had been described previously for KLF1 (37), in which KLF4 is fused to a tamoxifen-sensitive mutant of the estrogen receptor. The KLF4-ER fusion protein was subcloned into a lentivirus and used to transduce HL60 cells (see Materials and Methods). Because it had been demonstrated that KLF4 up-regulates p21 and down-regulates proliferation in other cell types (38), we selected and cellularly subcloned KLF4-ER-transduced HL60 clones which, in the absence of 4HT, expanded at rates similar to those of untransduced cells, to bias toward minimally leaky cell lines. Upon addition of 4HT to these selected KLF4-ER-transduced HL60 sub-cloned cell lines, proliferation slowed dramatically (Fig. 6A). PI staining 48 h after the addition of 4HT (but not vehicle controls) showed that KLF4-ER-induced cells arrested markedly in G₁ phase, as compared with transduction controls; the minor subdiploid population of PI-stained cells and absence of Annexin V-positive cells (data not shown) indicated that KLF4 expression did not greatly enhance apoptosis (Fig. 6B). Similar to previous reports in other cell types (38), we found that KLF4-induced cell cycle arrest was associated with increased levels of p21 (Fig. 6C), but not other cell cycle inhibitors. To assess the effects of induced KLF4 on monocytic differentiation, we immunostained KLF4-ER-transduced cells 48 h after addition of 4HT. The 4HT-treated cells (but not vehicle or transduction controls) expressed higher levels of the CD14 and CD11b monocytic markers (Fig. 6D), were non-adherent, and had monocytic morphology (Fig. 6, E and F).

KLF4 expression enhanced macrophage differentiation and blocked granulocytic differentiation of HL60 cells

We next examined the effects of conditional KLF4 expression on HL60 cells induced to differentiate into granulocytes or macrophages. HL60 cells differentiate into macrophages or granulocytes when cultured with TPA or RA, respectively. KLF4-ER transduced HL60 cells differentiated into monocytes in the presence of 4HT and macrophages in the presence of TPA (Fig. 7A), as expected. When we cultured KLF4-ER transduced HL60 cells in the presence of both TPA and 4HT, we observed a much more robust macrophage differentiation, with essentially every cell present in the culture contributing to a monolayer of macrophages (Fig. 7A, lower right). To assess granulocytic differentiation of HL60 cells, we measured the surface expression of CD66. When cultured in the presence of RA, a portion of the HL60 cells differentiate into granulocytes and up-regulate CD66. In contrast to the enhanced macrophage differentiation, granulocytic differentiation was blocked when KLF4-ER transduced cells were cultured with 4HT and RA (Fig. 7B). Interestingly, KLF4 induced monocytic differentiation was completely blocked when RA was present in the culture (Fig. 7B, lower right).