C57BL/6NRj mice from Janvier Labs (Le Genest-Saint-Isle, France) were bred at the Department of Comparative Medicine, Oslo University Hospital, Rikshospitalet (Oslo, Norway) in specific pathogen free conditions. The study was approved by the Norwegian National Committee for Animal Experiments. All experiments were performed in accordance with the institutional guidelines and regulations, including EU directive 2010/63/EU.

MOPC315 is a mineral-oil induced plasmacytoma cell line which was derived from a BALB/c mouse and was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) (38). Lewis lung carcinoma (LLC, also called LLC1) is a cell line originating from a spontaneous lung carcinoma in a C57BL/6 mouse and was obtained from CLS Cell Lines Service (Eppelheim, Germany) (39). L929 is a fibroblast-like cell line originating from connective tissue of a C3H/An mouse and was obtained from ATCC (40). J774.A1 is a macrophage-like cell line originating from the ascites of a BALB/c mouse with reticulum cell sarcoma and was kindly given by Anders Ø. Gammelsrud at the Norwegian Veterinary Institute (Oslo, Norway) (41). All of the above described cell lines were negative for mycoplasma infection as tested by use of MycoSensor PCR Assay kit (#302109, Agilent, Santa Clara, CA, USA).

Confluent L929 cells were cultured in RPMI 1640 medium (#61870044, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, #BCHRS0405, Biochrom GmbH, Berlin, Germany) for 10 days before the medium was centrifuged, filtered and stored at −20°C. Such L929 cell-conditioned medium contains macrophage colony-stimulating factor and was used for induction of macrophage differentiation according to standard protocols (42). Femurs and tibiae of the hind legs from 8- to 12-week-old male and female C57BL/6NRj mice were harvested and flushed with RPMI 1640 medium containing 10% FBS under sterile conditions. The isolated bone marrow was passed through a cell strainer with 70-µm pores (#CLS431751-50EA, Sigma-Aldrich, St. Louis, MO, USA) and cultured in 15 mm non-tissue culture treated dishes (#734-2359, VWR, Radnos, PA, USA) in RPMI 1640 medium containing 30% L929-derived conditioned medium. The bone marrow cells were cultured for 5 days, after which non-adherent, cells were washed off using phosphate buffered saline (PBS, # D8662, Sigma-Aldrich) and the adherent macrophages were cultured for 1 more day. Macrophages were harvested by incubation for 30 min at 4°C with cold PBS without CaCl₂ and MgCl₂ (#D8537, Sigma-Aldrich). Macrophages were then flushed off the plate, collected and kept frozen in aliquots at −150°C in FBS with 10% dimethyl sulfoxide (#0231-500 ml, VWR) for future experiments. The purity of the cells was 99% when analyzed by flow cytometry using the macrophage markers CD11b (#101219, BioLegend, San Diego, CA, USA) and F4/80 (#123116, BioLegend), and the cells were then referred to as bone marrow derived macrophages (BMDMs).

Bone marrow derived macrophages were thawed and cultured for 3 days in non-tissue culture treated dishes (VWR) in RPMI 1640 medium containing 10% FBS and 10% L929-derived conditioned medium. The BMDMs were harvested by scraping, incubated for 2 h at 37°C with 10 mg/ml mitomycin C (#M4287, Sigma-Aldrich) to inhibit proliferation, and then washed thoroughly. Next, the BMDMs were resuspended in medium and seeded out in triplicates in flat bottom 96 well plates (#734-1793, Costar, Washington, DC, USA) at 3 cell densities: 6 × 10⁴, 3 × 10⁴, and 3 × 10³ cells/well in a final volume of 200 μl/well. After 24 h of incubation, the medium was replaced with medium containing various stimuli, see section below, and incubated for another 24 h. Then, half of the cell supernatants (100 µl) were removed and used for quantification of NO₂⁻. Target cell suspensions, consisting of 5,000 cells/well of MOPC315 or 3,000 cells/well of LLC cells were added, resulting in varying ratios of effector to target cells. After 24 h of co-culture, cells were pulsed with [³H]thymidine (#MT6032, Hartmann Analytic, Braunschweig, Germany) and harvested 18 h later by a freeze and thaw cycle. The amount of radiolabeled DNA was measured on a 1450 MicroBeta Trilux Microplate Scintillation counter (Perkin Elmer, Waltham, MA, USA). The same assay was performed using the cell line J774.A1 as effector cells instead of BMDMs. Mitomycin C (10 mg/ml) was also used to block proliferation of J774.A1 cells before they were seeded out in 96 well plates in triplicates at a density of 1 × 10⁵ cells/well in a final volume of 200 μl/well.

For the purpose of statistical analysis of pooled data from several experiments, the percentage of remaining cancer cell growth was calculated by dividing counts per minute (cpm) values from the macrophage-LLC co-cultures at the 20:1 ratio with the cpm values of the corresponding wells with LLCs alone using the following equation: cpm20:1cpmLLC alone×100=% growth remaining.

The following TLR agonists were used: TLR1/TLR2 agonist Pam3CSK4 (Pam3, #tlrl-pms, InvivoGen, San Diego, CA, USA); TLR3 agonist poly(I:C) of high molecular weight type (#tlrl-pic, InvivoGen); TLR4 agonist LPS from E. coli (#L4391, Sigma-Aldrich); TLR5 agonist flagellin (FLA) from S. typhimurium, ultrapure type (#tlrl-epstfla-5, InvivoGen); TLR2/TLR6 agonist LTA from S. aureus (#L2515, Sigma-Aldrich); TLR7 agonist CL264 (#tlrl-c264e-5, InvivoGen); and TLR9 agonist CpG, class C ODN 2395 (#tlrl-2395-1, InvivoGen). The TLR agonists were used alone or in combination with mouse recombinant IFN-γ (#315-05, Peprotech, Rocky Hill, NJ, USA) at a 40 ng/ml concentration.

Supernatants were centrifuged at 410 g to remove cellular debris and immediately assayed for nitrite as a measure for the amount of NO that was produced. 50 µl of macrophage supernatant was added to 50 µl of Griess reagent A consisting of distilled water with 1% sulphanilamide (#S9251, Sigma-Aldrich) and 5% phosphoric acid (#W290017, Sigma-Aldrich). The mixture was incubated in the dark for 10 min. Next, 50 µl of Griess reagent B consisting of 0.1% N-(1-napthyl) ethylenediamine (#N9125, Sigma-Aldrich) in distilled water was added and the absorbance at 540 nm was measured with a microplate reader (BioTek Instruments, Winooski, VT, USA). Serial dilution of NaNO₂ served to create a standard curve of nitrite in the range of 1.56–100 µM.

S-Methylisothiourea hemisulfate salt (SMT, #M84445, Sigma-Aldrich) is a potent inhibitor of iNOS (43) which was used to block the production of NO by activated macrophages. Diethylenetriamine/NO adduct (DETA/NO) (#D185, Sigma-Aldrich) was used to produce controlled release of NO in solution.

Supernatants harvested from macrophages that had been stimulated with TLR agonists and/or IFN-γ for 24 h were centrifuged to remove cellular debris and stored at −80°C for maximum 1 week and assayed for cytokines. The cytokine concentrations were determined by multiplex bead assays, Bio-Plex Pro Mouse cytokine singleplex sets for TNF-α (#171-G5023M), IL-12p40 (#171-G5010M), IL-12p70 (#171-G5011M), monokine-induced by IFN-γ (MIG) (#171-G6005M), and IL-10 (#171-G5009M) from Bio-Rad Laboratories (Hercules, CA, USA) according to the manufacturer’s instructions. Samples in duplicates were analyzed, using a Bio-Plex MAGPIX Multiplex Reader and Bio-Plex Manager 6.1 software (Bio-Rad Laboratories).

Multiple groups were compared by using one-way ANOVA followed by a post hoc Tukey test for multiple comparisons and p values of less than 0.05 were considered statistically significant (*p-value < 0.05, **p-value < 0.01, ***p-value < 0.001). Statistical analysis, including column statistics, was performed using GraphPad Prism 7.02 software.

Table 1 shows an overview of the literature on induction of tumoricidal activity of macrophages by various TLR agonists. The most widely used agonist, LPS, has shown effect in a number of studies that utilized different functional assays, macrophages and target cells. LPS has been used alone, in combination with MAF/IFN-γ, other TLR agonists, agonistic anti-CD40 antibodies, or TGF-β inhibition. However, basic questions regarding the induction of tumoricidal activity in normal macrophages remain to be answered. Many of the studies from the 1970s and 1980s lacked reliably pure (LPS free) reagents or macrophages, and more recent articles typically lack functional assays for tumoricidal activity.

We used an in vitro growth inhibition assay (7, 13) in order to measure both the cytotoxic and cytostatic activity of macrophages toward tumor cells (Figure 1A). Macrophages were first treated with the DNA crosslinker mitomycin C to block cell division and thereby avoid that macrophage growth could interfere with the detection of tumor cell growth inhibition. Thereafter, macrophages were cultivated in the presence or absence of IFN-γ and TLR agonists for 24 h, before tumor cells were added and co-cultured for 42 h. Radiolabeled thymidine was used to detect tumor cell growth and was added to the co-cultures 18 h before cell harvest. As inhibition of tumor cell growth is known to depend on the number and density of macrophages, we seeded out macrophages in three different cell concentrations while the number of tumor cells remained constant within an experiment. The resulting ratio of macrophages to tumor cells, i.e., ratio of effector to target cells varied from 20:1 to 1:1 in various experiments.

In the first set of experiments, we investigated the effect of the classical macrophage activators IFN-γ and LPS. We used C57BL/6-derived BMDM as a source of normal mouse macrophages and the MOPC315 plasmacytoma as target tumor cells. When used alone, a high concentration (1,000 ng/ml) of LPS was required to activate BMDMs for inhibition of MOPC315 cell growth (Figure 1B). The potency of LPS was greatly improved when the macrophages were stimulated with LPS in combination with IFN-γ (Figure 1C) in accordance with previous reports (20, 22). Notably, macrophage stimulation with IFN-γ alone had no inhibitory effect on tumor cell growth (Figure 1C). Taken together, the experiments showed that activation with both LPS and IFN-γ was required for optimal induction of tumoricidal activity in macrophages. LPS alone could induce tumoricidal M1 macrophages, but only at high concentrations, while stimulation with IFN-γ alone had no effect.

To explore the potential of other natural or synthetic TLR agonists for inducing tumoricidal M1 macrophage phenotype, we tested a panel of agonists targeting different TLRs. In these experiments, the Lewis lung carcinoma (LLC) cell line was used as target cell line anticipating that macrophage-mediated tumor cell growth inhibition was not restricted to a single cell line. The target tumor cells were added to BMDMs activated by the following TLR agonists; TLR1/2 agonist Pam3, TLR2/6 agonist LTA, TLR3 agonist poly(I:C), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG (Figures 2A–F). Pam3 was very potent at stimulating the BMDMs and it resulted in strong growth inhibition of LLC cells, even at concentrations as low as 1 ng/ml, but only when it was used together with IFN-γ (Figure 2A). Similarly, IFN-γ in combination with LTA (Figure 2B), CL264 (Figure 2E), and CpG (Figure 2F) induced tumor cell growth inhibition by BMDMs. Stimulation of BMDMs with poly(I:C) resulted in growth inhibition both alone and together with IFN-γ. Similar to LPS, the effect of poly(I:C) was potentiated by IFN-γ (Figure 2C). Stimulation of BMDM with flagellin yielded no growth inhibition (Figure 2D). Statistical analysis was performed for two TLR agonists (Pam3 and CpG) by pooling data from experimental repeats. Because baseline cpm values varied between experiments, the percentage of growth remaining was used in the comparisons. The analysis revealed a statistically significant stronger growth inhibition when macrophages were activated by two signals (TLR agonist and IFN-γ) compared to one signal only (Figures 2G,H). Thus, induction of tumoricidal M1 macrophages can be achieved through stimulation of the TLRs 1/2, 2/6, 3, 4, 7, or 9 when combined with IFN-γ. Stimulation of TLR 3 and 4 has some effect alone at high ligand concentrations. The only TLR agonist tested that did not activate BMDMs was flagellin (TLR5).

To investigate whether our findings were of a more general value rather than being specific to BMDMs, we tested an immortalized murine macrophage cell line, J774A.1, in the growth inhibition assay. Mitomycin C was used to block J774A.1 cell growth, before stimulation with TLR agonists alone or in combination with IFN-γ. When activated by LPS and IFN-γ, the macrophage cell line induced very strong growth inhibition of MOPC315 cells (Figure 3). These results were consistent with the growth inhibition mediated by BMDMs (Figure 2). We observed similar effect of co-stimulation with IFN-γ and the agonists Pam3 and CpG for the J774.A1 cell line and the BMDMs, whereas the effect of poly(I:C) combined with IFN-γ was stronger for the cell line. IFN-γ and flagellin also successfully stimulated J774A.1 to inhibit growth. Thus, murine macrophages, either primary cells or a cell line, could be activated toward a tumoricidal M1 phenotype by stimulation with IFN-γ and a second signal. Several TLR agonists could provide this second signal.

Production of the cytotoxic free radical NO is considered a hallmark of M1-polarized pro-inflammatory macrophages (49). NO was shown to be crucial for macrophage-mediated defense against bacteria during normal immune responses (50) and has been reported to be important for the killing of tumor cells in vitro (51, 52). Due to the extremely short half-life of NO, we quantified it indirectly using the Griess assay. This assay is based on the Griess diazotization reaction of the NO metabolite nitrite (NO₂⁻) which forms a colored azo compound that can be quantified with a spectrophotometer. We analyzed the supernatant of BMDMs during the growth inhibition assay just before tumor cells were added. Macrophage activation with LPS alone for 24 h resulted in a concentration-dependent NO production (Figure 4A). Stimulation with LPS in combination with IFN-γ greatly potentiated the effect and yielded more than 10 µM NO₂⁻ already at the lowest concentration of LPS that was tested (0.1 ng/ml) (Figure 4A). At 1,000 ng/ml of LPS, there was no clear additive effect of co-stimulation with IFN-γ, and the NO₂⁻ production seemed to reach a maximum level around 15 µM. These results, where stimulation with IFN-γ greatly improved the effect of LPS, are in accordance with previous studies on NO induction (53). These data also support our finding in the growth inhibition assay, showing that stimulation with two signals is required for optimal induction of M1 macrophage phenotype, defined either by tumoricidal activity or NO production.

To investigate the importance of NO in macrophage-mediated tumor cell growth inhibition, we used the iNOS-specific inhibitor SMT to block NO production (43). SMT completely blocked NO production by activated BMDMs when used at 10 mM concentration, whereas 1 mM only partly hindered NO production (Figure 4B). When tested in the growth inhibition assay, 1 mM SMT was sufficient to abolish the growth inhibition induced both by LPS alone (Figure 4C) and by LPS in combination with IFN-γ (Figure 4D). These data strongly suggest that macrophages mediate growth inhibition of tumor cells through a NO-dependent mechanism.

To test whether we could recreate the growth inhibitory effect of NO without the presence of macrophages, we used the chemical compound diethylenetriamine/NO adduct (DETA/NO), which functions as an NO donor and releases NO to the medium. We set up a modified growth inhibition assay where tumor cells were exposed to DETA/NO in the absence of macrophages (Figure 5A). DETA/NO was dissolved in cell culture medium and used immediately. Just before adding the DETA/NO solution to the MOPC315 target cells, the amount of NO released in the medium was quantified indirectly by measuring NO₂⁻ for each concentration of DETA/NO used (Figure 5B). MOPC315 growth was then quantified by measuring incorporation of radiolabeled thymidine as for the standard growth inhibition assay (Figure 5C). We found that inhibition of growth was obtained only at the highest tested DETA/NO concentration, i.e., 1 mM, which corresponds to a NO₂⁻ concentration of around 100 µM (Figures 5A,B). These data confirm that our target tumor cells are sensitive to NO in a concentration-dependent way and are consistent with a key role of NO secretion for macrophage tumoricidal activity.

To examine whether TLR agonists other than LPS also induce NO production, we stimulated BMDMs with TLR agonists both alone and in combination with IFN-γ and measured the levels of NO₂⁻ in the supernatants (Figure 6A). For each TLR agonist, three concentrations were chosen and arbitrary defined as low, intermediate, and high. We found that all TLR agonists synergized with IFN-γ for induction of NO production, as the NO₂⁻ levels were 2- to 10-fold higher when BMDMs were activated with TLR agonists in combination with IFN-γ compared with TLR agonists alone. IFN-γ together with a low concentration of the TLR agonists still yielded more NO than single activation with TLR agonist at a 100-fold higher concentration (Figure 6A). Thus, several TLR agonists can replace LPS as an activating signal for macrophage NO production.

Next, we wanted to verify that the inhibitor SMT could inhibit the NO production induced by any TLR agonist. Measurements by the Griess assay revealed that SMT reduced the levels of NO₂⁻ in the BMDM cultures stimulated with TLR agonists in combination with IFN-γ (Figure 6B). Furthermore, we observed that the growth inhibition induced after co-stimulation with IFN-γ and LPS or any other tested TLR agonist was abolished when the iNOS inhibitor was present (Figure 6C). Therefore, in vitro tumor cell growth inhibition after macrophage activation with any TLR agonist appears to depend on NO production. Table 2 shows a summary of the effect of all tested TLR agonists in combination with IFN-γ. There was a strong, but incomplete correlation between induction of NO production and tumor cell growth inhibition.

In the next set of experiments, we wanted to examine whether release of particular cytokines was affected by single versus two signal activation of macrophages. We measured the levels of the pro-inflammatory cytokines TNF-α and IL-12p40, the Th1-polarizing cytokine IL-12p70, the anti-inflammatory cytokine IL-10, and the chemokine MIG/CXCL9 in the supernatant of BMDMs stimulated for 24 h (Figure 7). There was a clear synergistic effect of IFN-γ and most TLR agonists on the secretion of TNF-α, IL-12p40, and IL-12p70. Activation with TLR agonists alone resulted in relatively low to medium cytokine levels which increased in response to LPS, Pam3, LTA, and CL264 when IFN-γ was added (Figures 7A–C). IL-10 production was induced by TLR agonists alone with LPS giving the strongest response. Strikingly, IL-10 production induced by TLR triggering was reduced in the presence of IFN-γ (Figure 7D). Exceptions were poly(I:C) and flagellin, which resulted in no or very low secretion of any cytokine both when used alone and in combination with IFN-γ. Untreated BMDMs produced no cytokines. BMDMs activated with IFN-γ alone secreted no cytokines except for MIG/CXCL9 as expected for this IFN-γ-inducable chemokine. The chemokine MIG/CXCL9 was strongly induced by IFN-γ alone and the levels were further increased upon combined activation with all TLR agonists, except LPS (Figure 7E). Thus, IFN-γ and TLR agonists synergize to make macrophages produce high levels of pro-inflammatory and Th1-polarizing cytokines (TNF-α, IL-12p40, and IL-12p70) and low levels of IL-10.