Ionizing radiation reduces the capacity of activated macrophages to induce T-cell proliferation, but does not trigger dendritic cell-mediated non-targeted effects

Abstract

Purpose: Previous investigations revealed influences of irradiation up to 2Gy on the cytokine secretion profile of inflammatory and peritoneal mouse macrophages (pMФ). This raised the question if those alterations impact on dendritic cells and consecutive T-cell responses. Further, the impact of irradiation directly on pMФ capacity to induce T-cell responses was analyzed.

Materials and methods: pMФ were LPS-activated, irradiated and the expression of activation markers was assessed. Treated pMФ were co-incubated with T-cells to investigate proliferation. To verify modulating properties of pMФ supernatants isolated 24 h after irradiation, bone marrow-derived dendritic cells (BMDC) were co-incubated with supernatants and activation markers as well as the BMDC-induced proliferation of T-cells were measured.

Results: pMФ showed a highly significantly decreased major histocompatibility complexII (MHCII) expression within a dose range from 0.7–2Gy. Further, the proliferation rate of cluster of differentiation 4⁺ (CD4⁺) T-cells was decreased after co-incubation particularly with 2 Gy irradiated pMФ. The co-incubation of BMDC with supernatants of activated, irradiated pMФ significantly reduced the CD40 expression, but did not impact on the BMDC-derived induction of T-cell proliferation.

Conclusions: Inflammatory macrophages being exposed to irradiation have the potential to modulate consecutive adaptive immune reactions. But supernatants of irradiated macrophages do not influence the dendritic cells (DC)-mediated induction of T cell proliferation.

Keywords:

• Low-dose radiation
• activated macrophages
• dendritic cells
• non-targeted effects
• inflammation

Introduction

The induction of an inflammatory reaction is one of the key components of the body to deal with invading pathogens or tissue damage. Although serving as defense mechanism, an inflammatory reaction needs to be well regulated. If this is not the case an uncontrolled inflammation and related damage pathways could give rise to benign and malignant diseases (reviewed in Wunderlich et al. (2017)).

One key player in the induction and resolution of inflammatory processes is the macrophage. This highly distributed innate immune cell clears the body from pathogens, dying cells, cellular debris and additionally influences the recruitment and function of other immune cell types such as dendritic cells (DC) by the secretion of cytokines and chemokines (Unanue et al. 1976; Medzhitov and Janeway 1997; Gordon and Taylor 2005). Further, macrophages have the potential to act as antigen presenting cell (APC) and induce an adaptive T-cell immune reaction (Pozzi et al. 2005; Galli et al. 2011). Today it is well accepted that macrophages can display different phenotypes influenced by their micromilieu (Gordon and Taylor 2005). Different phenotypes such as classically activated macrophages (M1), wound-healing macrophages (M2) and regulatory macrophages can be e.g. distinguished according to their cytokine and chemokine expression profile (Mantovani et al. 2002). M1-macrophages display a pro-inflammatory phenotype upon toll-like receptor (TLR) activation with e.g. bacterial compounds like lipopolysaccharide (LPS) and secrete pro-inflammatory cytokines like tumor necrosis factor alpha (TNFα) or interleukin-1beta (IL-1ß; Mosser and Edwards 2008). Additionally, the phenotype can be induced by interferon-gamma (IFN-γ) derived from other immune cells like natural killer (NK) cells (Dale et al. 2008). In contrast, the M2-macrophages are associated with wound-healing and can be induced by IL-4 or IL-13 derived from other immune cells (Stein et al. 1992). Similar to M2-macrophages, the third subtype is a regulatory subtype to which tumor associated macrophages (TAM) belong (Mosser and Edwards 2008). The induction of this subtype is supposed to be induced by tumor-derived factors such as prostaglandins or by tissue conditions such as hypoxia (Knowles and Harris 2001; Kuang et al. 2007).

Due to their pivotal functions in inflammation and their plasticity in changing the phenotype upon the micromilieu, the contribution of macrophages to the initiation, progression and therapy outcome of various diseases is of special interest.

In terms of benign, inflammatory diseases like rheumatoid arthritis (RA) it is getting more and more clear that an increased number of inflammatory macrophages located within the inflammatory site can contribute to the sustained or worsened course of disease by the fine balanced secretion of pro- and anti-inflammatory cytokines (Mulherin et al. 1996; Miossec and van den Berg 1997; Tak et al. 1997; Kinne et al. 2000; Haringman et al. 2005). The macrophage-derived cytokine milieu can induce an inflammatory T-cell response by inducing the T_H17 T-cell subtype (McInnes and Schett 2011).

Beside the direct negative effects of the inflammatory disease (pain, swelling, etc.), patients suffering e.g. from Crohn´s disease are associated with a higher risk of developing malignant colorectal diseases (Hamilton 1985; Brostrom et al. 1987). The fact that inflammation can support carcinogenesis has led to the definition of a tumor-promoting inflammation as an enabling characteristic of cancer (Hanahan and Weinberg 2011). As for benign diseases, macrophages located in near proximity to the developing tumor can contribute to a tumor-promoting inflammation. Representing a double-edged sword, M1-macrophages mainly located in developing tumors can act in an anti-tumor manner but also support the carcinogenesis by the secretion of cytokines inducing angiogenesis and genomic instability (Murdoch et al. 2008; Zumsteg and Christofori 2009; Qian and Pollard 2010; Ong et al. 2012). Nevertheless, during tumor progression the M1 phenotype switches into a more anti-inflammatory regulatory TAM, which also influences the tumor cell proliferation, angiogenesis and suppression of anti-tumor-immunity by the secretion of cytokines and other factors (Wyckoff et al. 2004; Guruvayoorappan 2008; Coffelt et al. 2009).

Inflammatory as well as malignant diseases are treated with radiotherapy (RT) since a long time, but with different single dose. In the treatment of malignancies, a single high dose of around 2Gy is used in a fractionated schedule to induce stop of tumor cell proliferation or tumor cell death and is attributed with an induction of inflammation. Low-dose radiotherapy (LD-RT, single dose ≤1 Gy) is used to treat inflammatory diseases and an amelioration of inflammation due to the LD-RT is supported by pre-clinical and clinical studies (Keilholz et al. 1998; Glatzel et al. 2004; Ruppert et al. 2004; Ott et al. 2014; Frey et al. 2015, Reichl et al. 2015; Rodel et al. 2017). A recent study by Ruhle et al. (2016, 2017) showed a significant pain relief in patients with different inflammatory and degenerative disease pattern due to low dose radon spa therapy. The clinical improvement was linked to modulations of the global peripheral blood immune cell composition and in particular correlated with reduced expression of activation markers on immune cells.

Looking more mechanistically on tissue-specific immune cells, the anti-inflammatory effect is in part linked to a selectin-chemokine-mediated reduced evasion of immune cells from the blood and an increased apoptosis in peripheral blood mononuclear cells in a discontinuous dose-dependent manner, especially in a dose range from 0.3 to 0.7 Gy (Kern et al. 1999, 2000; Hildebrandt et al. 2002; Rodel et al. 2008). Further, several influences of LD-RT on inflammatory macrophages were detected. For instance, the oxidative burst was altered by a reduction of reactive oxygen species (ROS) and a reduced nitrite oxide production (NO) related to a suppressed activation of the inducible nitrite oxide synthase (iNOS) in RAW 264.7 macrophages (Hildebrandt et al. 2003a, 2003b). In addition, a nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), extracellular-signal-regulated kinase 1/2 (ERK 1/2) and mitogen activated protein kinase (MAPK) p38 dependent decreased secretion of inflammatory cytokines such as IL-1ß or TNFα was detected after treatment of human and mouse macrophages with in particular 0.5 or 0.7 Gy of X-ray (Lodermann et al. 2012; Tsukimoto et al. 2009). Of note is that the impact of ionizing radiation applied in LD-RT on the inflammatory phenotype of activated macrophages is dependent on the basal radiosensitivity (Frischholz et al. 2013). Functional analyses like chemotaxis and migration experiments revealed that a reduced migration, but an induced chemotaxis of inflammatory peritoneal mouse macrophages after X-ray treatment with 0.5 Gy could additionally contribute to amelioration of inflammation (Wunderlich et al. 2015). Furthermore, no influence on the phagocytosis and viability were detected, what is a positive result related to radiation protection issues, as macrophages play pivotal roles in the clearance of dying and damaged cells and thereby preventing the induction of auto-immune diseases (Gaipl et al. 2006; Conrad et al. 2009; Wunderlich et al. 2015).

Beside the direct harmful effects of irradiation on cellular components of the exposed cell (targeted effect) like deoxyribonucleic acid (DNA), membranes and enzymes, also a phenomenon called non-targeted or bystander effect can occur in cells not being exposed to irradiation. These cell type-, radiation-dose- and radiation-type-dependent effects include increased DNA-damage, increased/decreased clonogenic survival or apoptosis (Lehnert et al. 1997; Mothersill and Seymour 1997; Prise et al. 1998; Sokolov et al. 2005; Rzeszowska-Wolny et al. 2009). The effects are supposed to be mediated by released or directly transmitted factors like long-lived ROS or cytokines from and between irradiated and non-irradiated cells (Narayanan et al. 1999; Iyer et al. 2000; Harada et al. 2008).

The above-mentioned facts made clear that radiation in a low dose range is capable of modulating the macrophages inflammatory status, especially regarding the cytokine secretion profile. Nevertheless, other immune cells, like DC are also involved in inflammatory processes within tissues. Particularly DC are regarded as the bridging cell component between innate and adaptive immunity steering systemic immune reactions by acting as APC and inducing T-cell responses (Steinman 1991; Banchereau and Steinman 1998). As DC can be located in near proximity of macrophages during inflammatory reactions, we were interested in how and to what extend bystander effects could be induced in DC by the above-mentioned modulations in macrophages cytokine secretion triggered by low dose irradiation.

We therefore investigated if supernatants (SN) of ex-vivo activated, irradiated peritoneal mouse macrophages (pMФ) have the capacity to modulate the surface expression of activation markers on syngeneic bone marrow-derived dendritic cells (BMDC) and to impact on the BMDC-related induction of proliferation in allogeneic T-cells within an inflammatory microenvironment. In addition, we analyzed the direct impact of irradiation on the expression of activation-related surface molecules on inflammatory pMФ and lastly macrophage-mediated non-targeted effects on allogeneic T-cell proliferation.

Material and methods

Mice for the isolation of the different immune cell types

All mice used for extraction of pMФ, BMDC and allogeneic T-cells were bred under sterile atmosphere at the animal facility of the Friedrich-Alexander-Universität Erlangen-Nürnberg (Franz-Penzoldt-Centre). BALB/c mice used for the extraction of pMФ were aged between 30 and 35 weeks and age-controlled for the respective experiments. Further, BALB/c mice used for the extraction of bone marrow to generate BMDC were aged between six and 10 weeks and age-controlled for the respective experiments. Lastly, the spleen of C57/BL6 mice was isolated at an age of 6–10 weeks to generate allogeneic T-cells for mixed lymphocyte reaction (MLR). The animal procedures have been approved by the ‘Regierung of Mittelfranken’ and were conducted in accordance with the guidelines of Federation of European Laboratory Animal Science Associations (FELASA).

Generation and isolation of peritoneal macrophages

pMФ were generated by injection of 2.5 ml of 4% (w/v) Brewer`s thioglycollate broth into the peritoneal cavity of mice as previously described by Schleicher and Bogdan (2009). Isolation of pMФ was performed four days after the injection by washing the peritoneum with warm phosphate-buffer saline (PBS). Subsequently, cells were seeded at a density of 2 x 10⁶ per well in low-adherence suspension six-well plates using R10 (RPMI 1640 with stable glutamine, supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum) media and incubated for at least 4 h at 37 °C, 5% CO₂ and 90% humidity prior to treatment, allowing them to properly adhere to the plastic.

Treatment of peritoneal mouse macrophages

After seeding and incubation, cells were activated and treated with X-ray according to Wunderlich et al. (2015). In brief, pMФ were either activated with 1 μg/ml LPS or left non-activated as control for 16 h at 37 °C, 5% CO₂ and 90% humidity. Consecutively, activated pMФ were irradiated with a single dose up to 2Gy (0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 1.0 or 2.0 Gy) with a X-ray generator (100 kV; GE Inspection Technologies, Hürth, Germany). Afterwards, pMФ were cultivated for 24 h before performing the analysis.

Isolation of supernatants of activated, irradiated peritoneal mouse macrophages

We previously showed that irradiation impacts on the cytokine secretion of activated peritoneal macrophages (Wunderlich et al. 2015). We therefore were now interested in how these modulations could have impact on the function of DC and especially on the expression of surface molecules and their capacity to induce proliferation in naïve T-cells. Therefore, we collected the SN of differently treated macrophages 24 h after irradiation, centrifuged the SN to get rid of cellular components and then used the SN for co-activation experiments with BMDC.

Analyses of activation markers of activated, irradiated pMФ

To analyze the surface expression of activation markers of pMФ after irradiation, cells were isolated from the cell culture material using a cell scraper and by rinsing the well with ice-cold PBS. Less adherent cells within the SN were combined with the adherent fraction to avoid non-adherent cells being lost for the analyses. The cell suspension was co-incubated with 5 μg/ml cluster of differentiation (CD) 16/32 antibody (eBioscience, San Diego, CA) to avoid unspecific FcγIII/FcγII-receptor antibody binding and subsequently stained with F4-80, CD11b, major histocompatibility complex (MHC) II (all eBioscience), CD80 and CD86 (all BD Pharmigen, New York, NY) antibodies.

After 30 min incubation, the surface expression was determined using flow cytometry, whereas all measurements were performed in quadruplicates. The macrophage fraction was determined by the surface expression of CD11b and F4-80. The expression of all other surface markers was related to the CD11b⁺/F4-80⁺ cell population. Expression of the molecules was related to the mean fluorescence intensity (MFI).

Generation of bone marrow-derived dendritic cells of BALB/c mice

Isolation and generation of BMDC was performed according to Lutz et al. (1999). In brief, 2 x 10⁶ bone marrow derived cells isolated from femur and tibia of the hind legs of BALB/c mice were seeded in 100 x 15mm petri dishes (BD Falcon, New York, NY). The generation of BMDC was performed using DC-Media (RPMI 1640 with stable glutamine, supplemented with 1% penicillin-streptomycin, 0.1% ß-Mercaptoethanol and 0.004 μg/ml mouse GM-CSF). The cells were cultivated for seven days at 37 °C, 5% CO₂ and 90% humidity, whereas DC-media was added at day 3 and 6 post isolation. At day 7, BMDC were collected and used for experiments.

Analyses of surface expression of activation markers on bone marrow-derived dendritic cells after co-incubation with supernatants of activated, irradiated pMФ

Co-incubation was performed by seeding 1 x 10⁶ BMDC in six-well plates. Subsequently, the cells were incubated with SN of activated, irradiated pMФ for 16 h at 37 °C, 5% CO₂ and 90% humidity. Incubation of BMDC with pMФ culture media, SN of non-activated, non-irradiated pMФ and BMDC activated with 1 µg/ml LPS served as controls. After the 16 h incubation period, the co-activated BMDC were isolated and stained with 5 µg/ml CD16/32 antibody (eBioscience) to avoid unspecific FcγIII/FcγII-receptor antibody binding. Subsequently, cells were stained with antibodies against CD11c, CD40, CD80, CD83, CD86 and MHCII for 30 min at 4 °C followed by analysis by flow cytometry. The BMDC fraction was determined according to the surface expression of CD11c and MHCII. The expression of the surface markers CD40, CD80, CD83 and CD86 was always related to the CD11c⁺/MHCII⁺ cell fraction. Expression of the molecules was related to the MFI.

Isolation of splenic T-cells from C57/BL6 mice by negative selection

Negative selection of T-cells was performed using the magnetic separation kit mouse Pan T-Cell Isolation Kit II (Miltenyi Biotek, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. In brief, splenic cells were stained with an antibody cocktail against surface markers of non T-cell cells (CD11b, CD11c, CD19, CD45R, CD49b, CD105, Anti-MHCII and Ter-119) for 10 min at 4 °C. Subsequently, the cell suspension was incubated for 15 min at 4 °C with magnetic-labeled anti-biotin microbeads. After a washing step, the magnetic separation was performed using Column MS columns (Miltenyi Biotek, Bergisch Gladbach, Germany). The quality of each separation was verified by a staining of the cell suspension with CD3, CD4 and CD8 antibodies and consecutive analyses by flow cytometry. After the separation, the isolated T-cell suspension was stained with carboxyfluorescein succinimidyl ester (CFSE) to analyze T-cell proliferation (Lyons and Parish 1994).

Mixed lymphocyte reaction using bone marrow derived dendritic cells or activated, irradiated pMФ

The MLR was performed using co-activated BMDC or activated, irradiated pMФ from BALB/c mice, respectively (as described above). 2.5 x 10⁴ of either BMDC or pMФ were co-incubated with 5 x 10⁴ CFSE stained T-cells from allogeneic C57/BL6 mice in U-bottom shaped culture plates (BD Pharmigen) at 37 °C, 5% CO₂ and 90% humidity for five days. After co-culturing, cells were isolated, incubated with 5 μg/ml CD16/32 antibody (eBioscience) to avoid unspecific FcγIII/FcγII-receptor antibody binding and further stained with CD3, CD4, CD8 and CD25 antibodies (30 min, 4 °C). Consecutively, proliferation was analyzed by flow cytometry. T-cell proliferation was related to a reduced fluorescence signal of CFSE compared to non-proliferated controls, whereas CFSE fluorescence was merely referred to the CD4⁺ or CD8⁺ T-cell fraction, respectively. In addition, CD25 surface expression related to the CD4+ and CD8+ cell populations was analyzed, whereas the expression was related to the MFI. T-cells incubated without BMDC or pMФ served as controls. Supplementary Figure 1 displays the gating strategy for these analyses.

Figure 1. Overview of the experimental setting for the analyses of activation status of irradiated peritoneal mouse macrophages (pMФ), co-activated bone marrow-derived dendritic cells (BMDC) and induced proliferation in allogeneic naïve T-cells exhibited by pMФ and BMDC: subsequent to activation, irradiation, and incubation of pMФ, several analyses and co-activation experiments were performed. First, the influence of irradiation on the surface expression of activation markers on activated pMФ was analysed (1). Further, the potential of activated, irradiated pMФ to induce proliferation in carboxyfluorescein succininimidyl ester (CFSE)-stained, allogeneic naïve T-cells was verified by co-incubation of both cell types for five days and consecutive analyses by flow cytometry (2). Additionally, the supernatants of activated, irradiated pMФ were isolated and transferred on syngeneic immature bone marrow-derived dendritic cells (BMDC) (3, 4). After co-incubation for 16 h, the expression of activation markers on co-activated BMDC was determined by flow cytometry (5). Finally, co-activated BMDC were incubated for five days with CFSE-stained, allogeneic naïve T-cells and the proliferation rate was determined using flow cytometry (6).

Statistical analysis

Figure 1 summarizes the experimental settings for the above described analyses. pMФ of at least four individual mice were used for surface expression analysis and MLR experiments. Further, the SN from irradiated pMФ of at least four mice were used for the co-activation experiments with BMDC with respect to surface analysis and MLR. Statistical analysis were performed using unpaired student`s t-tests. Additionally, normal distribution was verified by Shapiro-Wilk normality test. Results were considered statistically significant for p < .05 (★) and highly significant for p < .01 (#).

Results

Irradiation impacts on the surface expression of MHCII on activated pMФ

No significant modulations of the surface expression of the activation markers CD80 and CD86 on activated pMФ was observed after irradiation with single doses of ionizing radiation up to 2Gy (Supplementary Figure 2). In contrast, irradiation with a single dose starting at 0.3 Gy and up to 2 Gy significantly and starting from 0.7 Gy highly significantly reduced the surface expression of MHCII (basal expression with a MFI of about 4; Supplemenary Figure 3(A)) on activated pMФ (Figure 2(A)).

Figure 2. MHCII surface expression, CD4⁺ T-cell proliferation and CD25 expression on CD4⁺ T-cells in dependence of peritoneal mouse macrophages (pMФ)with different activation and irradiation status: subsequent to activation, irradiation with indicated dose and incubation for 24 h, pMФ were isolated and surface major histocompatibility complex II (MHCII) expression was analyzed by flow cytometry (A). The expression of MHCII was only related to the cluster of differentiation (CD) 11b⁺/F4-80⁺ cell fraction. Further, the treated pMФ were co-incubated with carboxyfluorescein succininimidyl ester (CFSE)-stained, allogeneic, naïve T-cells for five days and proliferation rate of CD4⁺ T-cells (B) and the CD25 surface expression (C) by CD4⁺ T-cells was assessed by flow cytometry. Each graph shows the fold change of four individual experiments, whereas the value of activated, non-irradiated pMФ (0.0 Gy) was set to one and all other values were referred to it. Error bars show standard deviation. ^★p < .05, ^#p <.01; calculated with the unpaired student´s t-test against 0 Gy, normal distribution of samples was verified using Shapiro-Wilk normality test. o/o: non-activated and non-irradiated pMФ.

Figure 3. Activation status of bone marrow-derived dendritic cells incubated with supernatants of activated, irradiated peritoneal mouse macrophages(pMФ): subsequent to activation, irradiation with indicated dose and incubation for 24 h, the supernatants (SN) of pMФ were isolated and transferred on immature bone marrow-derived dendritic cells (BMDC). After co-incubation for 16 h, the expression of surface activation markers cluster of differentiation (CD) 80 (A), CD86 (B), CD40 (C) and CD83 (D) on BMDC was analyzed by flow cytometry. The expression of the different markers was only related to the CD11c⁺/MHCII⁺ cell fraction. Each graph shows the fold change of four individual experiments, whereas the value of the activated, non-irradiated pMФ was set to one and all other values were referred to it. Error bars show standard deviation. ^#p < .01; calculated with the unpaired student´s t-test against 0 Gy. Normal distribution of samples was verified using Shapiro-Wilk normality test. o/o: SN of non-activated and non-irradiated pMФ; DC LPS: DC incubated with 1 µg/ml lipopolysaccharide; DC media: DC incubated with macrophages media.

Irradiation impacts on the capacity of activated pMФ to activate T-cells

The MLR experiments revealed an influence of irradiation on the capacity of pMФ to induce proliferation in allogeneic CD4⁺ T-cells displaying a basal proliferation of about 60% (Supplementary Figure 3(B)). The proliferation of CD4⁺ T-cells decreased when the T cells were incubated with previously irradiated pMФ, whereas only a single dose of 2 Gy reduced the induction of proliferation of T cells by macrophages significantly (Figure 2(B)). In addition, the expression of the CD25 activation marker on T-cells (basal expression with a MFI of about 2.5; Supplementary Figure 3(C)) was significantly reduced after co-incubation of the T cells with macrophages that had been exposed to a single dose of 0.01, 1 and 2 Gy, respectively in comparison to activated, non-irradiated pMФ (Figure 2(C)). Further, also the co-incubation of T-cells with non-activated and non-irradiated (o/o) pMФ also significantly reduced the surface expression of CD25 compared to the activated and non-irradiated ones.

Supernatants of irradiated, activated pMФ modulate the surface expression of CD40 on BMDC

The co-incubation of BMDC with SN of irradiated, activated pMФ did not significantly impact on the expression of the classical co-stimulatory molecules CD80 and CD86 (Figure 3(A,B)), with a basal expression with a MFI of about 45 for CD80 and 63 for CD86, respectively; Supplementary Figure 4(A,B)). However, surface expression of CD40 (basal expression with a MFI of about 28; Supplemenatry Figure 4(C)) was significantly reduced on BMDC after co-incubation with the SN of macrophages that had been exposed to irradiation, even with very low doses (0.01 Gy). In addition, the co-incubation with DC media control or the incubation with SN of non-irradiatedand non-activated pMФ also reduced the CD40 surface expression in a similar manner (Figure 3(C)). The expression of the prominent DC activation marker CD83 (basal expression with a MFI of about 3.8; Supplemenatry Figure 4(D)) was slightly, but not significantly reduced in particular after co-incubation of BMDC with SN of activated pMФ that had been irradiated with 0.3, 1.0 or 2.0 Gy, respectively (Figure 3(D)).

Figure 4. Proliferation of CD4⁺ or CD8⁺ T-cells and related CD25 expression after co-incubation with bone marrow-derived dendritic cells co-cultured with supernatants of activated and irradiated peritoneal mouse macrophages (pMФ): the supernatants (SN) of activated pMФ were isolated 24 h after irradiation and subsequently co-incubation of bone marrow-derived dendritic cells (BMDC) with the SN was performed for 16 h. Consecutively, co-activated BMDC were co-cultured with carboxyfluorescein succininimidyl ester (CFSE)-stained, allogeneic naïve T-cells. After five days the proliferation of cluster of differentiation (CD) 4⁺ (A) as well as CD8⁺ (B) T-cells and related CD25 expression (C,D) was determined using flow cytometry. Each graph shows the fold change of four individual experiments, whereas the values of DC co-incubated with supernatants from activated, non-irradiated pMФ were set to one and all other values were referred to it. Error bars show standard deviation. o/o: DC co-incubated with SN of non-activated and non-irradiated pMФ; DC LPS:DC incubated with 1 µg/ml lipopolysaccharide; DC Media: DC incubated with macrophages media.

Supernatants of irradiated, activated pMФ do not impact on the capacity of BMDC to induce T-cell proliferation

The incubation of BMDC with SN of irradiated, activated pMФ was, however, without impact on the capacity of BMDC to induce proliferation in allogeneic CD4⁺ T cells (basal proliferation of about 50%; see Supplementary Figure 5(A)) and CD8⁺ T-cells (basal proliferation of about 80%; Supplementary Figure 5(A)), respectively. Furthermore, no significant influence on the CD25 surface expression on allogeneic T-cells was detected (Figure 4(C,D), with CD25 displaying very low basal expression with a MFI of about 1 (Supplementary Figure 5(C,D)).

Discussion

Macrophages as key players in the induction and resolution of inflammation are modulated in their cytokine secretion, chemotaxis and transmigration by irradiation up to 2Gy in an anti-inflammatory manner, especially after irradiation with 0.5 or 0.7 Gy (Lodermann et al. 2012; Frischholz et al. 2013; Wunderlich et al. 2015). These observations lead to the assumption that also the expression of activation makers as well as the related capacity of macrophages to induce T-cell proliferation could be altered by radiation. Our experiments revealed an impact of irradiation up to 2.0 Gy on the surface expression of MHCII. In particular irradiation with 0.7, 1.0 or 2.0 Gy resulted in highly significant reduced expression of MHCII (Figure 2(A)). This goes along with a highly significant reduced translocation of NFκB p65 into the nucleus of irradiated inflammatory pMФ (Lee et al. 2006; Yang et al. 2008; Wunderlich et al. 2015,). Since NFκB regulates many genes that are involved in inflammatory processes and immune regulations, several other pathways most likely contribute to the here described alterations of the phenotype of the activated macrophages following exposure to radiation (Diamant and Dikstein 2013). Radiation-induced cell death does not impact on it, since no major induction of cell death in macrophages by irradiation up to 2 Gy was already shown (Wunderlich et al. 2015).

Moreover, macrophages have the capacity to act as APC. As MHCII and co-stimulatory molecules such CD80 or CD86 are involved in T-cell activation, influences of the altered MHCII expression on the induction of T-cell proliferation by macrophages could be assumed (Shortman and Liu 2002; Pozzi et al. 2005; Galli et al. 2011). Even though MHCII expression was significantly down-regulated, the consecutive effects on reduced T-cell proliferation and reduced expression of the activation marker CD25 on T-cells were less pronounced (Figure 2(B,C)). This calls for additional factors which are involved in T-cell stimulation such as further co-stimulatory molecules and soluble factors such as cytokines (Smith-Garvin et al. 2009; Davoodzadeh Gholami et al. 2017). The impact of radiation on these factors under inflammatory conditions is part of ongoing and future research (Frey et al. 2015).

We conclude that activated macrophages have the potential to induce non-targeted effects in T-cells and thereby might alter the adaptive immunological response after single dose irradiation up to 2 Gy. In vivo migration of macrophages into lymph nodes or the localization of macrophages in lymph nodes has already been described. Here alterations of a systemic immunological response via irradiated macrophages could take place (Bellingan et al. 1996; Cao et al. 2005; Haringman et al. 2005). The modulation of T-cell proliferation by irradiated macrophages could also have impact on benign and malignant diseases and related therapy regimes. Cancer therapy often includes irradiation schemes of tumors and related lymph nodes with a single dose of approximately 2Gy. As the tumor microenvironment includes a variety of immune cells and the latter are located within potentially irradiated lymph nodes, the influence of irradiation on cells of the immune system and their functionality, in addition to the mere impact on tumor cells, should be considered in multimodal cancer therapies (Frey et al. 2016). It has already become evident that cancer therapy outcome is in part related to immunological parameters such as the infiltration of T-cell subpopulations into the tumor (Balermpas et al. 2016). How the modulations in the induction of an adaptive immune response triggered by irradiated macrophages could have impact on the anti-tumor immunity remains elusive and should be investigated in future experiments.

Especially LD-RT is used to treat benign, inflammatory diseases using a dose range from 0.5–1.0 Gy. Although the proliferation of CD4⁺ T-cells was not reduced significantly at these single doses (Figure 2(B)), even a slightly reduced proliferation rate could contribute to the well-described anti-inflammatory effect of LD-RT in a dose range from 0.5–1.0 Gy (summarized in (Rodel et al. 2012)). RA for example is an inflammatory disease in part mediated by T_H1 and T_H17 cells (reviewed in (McInnes and Schett 2011)). Therefore, a reduced macrophage-derived CD4⁺ T-cell proliferation could also reduce the amount of disease-related T_H1 and T_H17, as the latter two are both a subtype of the CD4⁺ T-cell subpopulation. Thereby a lower amount of T_H1 and T_H17 cells could ameliorate the inflammatory reaction.

In addition to macrophages, DC can be located within inflammatory sites and thereby could be influenced through other immune cells such as macrophages. As DC are professional APC, they bridge the innate and adaptive arm of the immune system, what makes them a key cell in the induction of a systemic immune response (Steinman 1991). Therefore, we were interested in how the modulated macrophage-derived cytokine profile after a single dose irradiation of activated macrophages of up to 2Gy could induce bystander effects in non-irradiated DC (schematic overview is presented in Figure 1). A special emphasis was set on possible modulations on the expression of surface markers and the DC-related induction of T-cell proliferation. Except for CD40 (Figure 3(C)), no significant alterations of the activation marker expression on BMDC were detected. The reduced CD40 surface expression could lead to a reduced T-cell proliferation, as the ligation of CD40 on DC with its receptor CD40 Ligand on T-cells leads to a fully induced antigen presenting function of DC, what in reverse impacts back on the T-cell proliferation (van Kooten and Banchereau 2000; Ma and Clark 2009). Nevertheless, no significant influence on the proliferation of neither CD4⁺ nor CD8⁺ T-cells was detected (Figure 4). These findings suggest that modulations in macrophages exhibited by irradiation up to 2 Gy do not impact on the DC capability to induce an adaptive T-cell response, what is a very positive result with regard to radiation protection issues. Furthermore, in regard of diseases therapy, the DC-mediated T-cell response is maintained (Deloch et al. 2016). Induction of anti-tumor immunity during treatment of malignancies is maintained, as DC can still take up tumor specific antigens, present them and induce the proliferation of tumor specific T-cells (Preynat-Seauve et al. 2006). Nonetheless, the sustained induction of proliferation of T-cells could also have negative effects in the light of benign, inflammatory diseases as the proliferation of the disease-related T-cells (T_H1 and T_H17) is perpetuated.

In summary, irradiation of activated (inflammatory) macrophages has the potential to induce bystander effects in DC and in T-cells. Future research should clarify if also abscopal effects, describing radiation-induced responses in non-irradiated tissues, are induced under these conditions, as already seen after therapy of malignant diseases with combination of radiotherapy and immunotherapy (Sham 1995; Ohba et al. 1998; Wersall et al. 2006; Postow et al. 2012). Macrophages being exposed to irradiation and acting as APC have the potential to modulate consecutive immune reactions in T-cells under inflammatory conditions. But alterations in the cytokine profile of irradiated macrophages do not influence the DC-mediated induction of proliferation in T-cells.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the European Commission [DoReMi, European Network of Excellence, contract number 249689] and German Federal Ministry of Education and Research [GREWIS, 02NUK017G].

Notes on contributors

Roland Wunderlich

Roland Wunderlich focused his PhD work within the European Network of Excellence DoReMi on immune effects of low doses of ionizing radiation on activated immune cells. He also worked as postdoc on non-targeted effects of ionizing radiation with focus on tumor immunology. He currently works as product specialist for flow cytometry research.

Paul Friedrich Rühle

Paul-Friedrich Rühle worked as a PhD student and a postdoc on the establishment of detailed immunophenotyping protocols for patients who had been exposed to radiation for therapy reasons. His special interests are systemic immune effects following local radiation exposure.

Lisa Deloch

Lisa Deloch research interests are osteoimmunological effects of low and intermediate doses of radiation with different qualities (X-rays and alpha-particles). Her special focus is set on the impact of the basal inflammatory status on the radiation response.

Franz Rödel

Franz Rödel is head of the Radiation Biology at the Department of Radiotherapy and Oncology at the Goethe-University Frankfurt am Main. He has focused on the prognostic and predictive impact of tumor infiltrating immune cells as well as on immunological mechanisms of low doses of ionizing radiation.

Rainer Fietkau

Rainer Fietkau is director of the Department of Radiation Oncology of the Universitätsklinikum Erlangen (UKER). The department offers the entire spectrum of modern radiotherapy including low dose radiotherapy for benign diseases and multimodal radiooncological therapies from one source. Clinical aspects of radiation oncology are examined within phase I, II, and III trials.

Udo S. Gaipl

Udo S. Gaipl is the head of the Radiation Immunobiology Group at UKER. He is the leading scientist in the field of how stressed cells alone and in interaction with innate and adaptive immune cells modulate the immune system. Immune biomarkers are followed up to predict radiation-induced and/or –modulated inflammatory responses.

Benjamin Frey

Benjamin Frey is Lab-Manager, Study-Coordinator and Deputy Head of the Radiation Immunobiology Group at UKER. His research also focuses on immune modulation by low, intermediate and high doses of radiation. He is an expert in pre-clinical animal models for radiation responses and in coordination of studies that follow-up immune changes in patients during radiotherapy of benign and malign diseases.