Inflammatory Signaling and DNA Damage Responses after Local Exposure to an Insoluble Radioactive Microparticle

Simple Summary A cesium-bearing microparticle (Cs-BMP) is an insoluble radioactive microparticle possessing high specific radioactivity, which was discovered after the incident at the Fukushima nuclear power plant. Due to their insoluble nature, such Cs-BMPs are assumed to adhere in the long term to normal tissue, leading to chronic local exposure. However, radiation risk due to the intake of internal exposure to radioactive cesium is conventionally estimated from the organ dose given by uniform exposure to soluble cesium. As such, it is critical to clarify the normal tissue effects posed by heterogeneous exposure to Cs-BMPs. This in vitro study reports on the relationship between the inflammatory responses and DNA damage induction during local exposure to a Cs-BMP. Abstract Cesium-bearing microparticles (Cs-BMPs) can reach the human respiratory system after inhalation, resulting in chronic local internal exposure. We previously investigated the spatial distribution of DNA damage induced in areas around a Cs-BMP; however, the biological impacts have not been fully clarified due to the limited amount of data. Here, we investigated the inflammatory signaling and DNA damage responses after local exposure to a Cs-BMP in vitro. We used two normal human lung cell lines, i.e., lung fibroblast cells (WI-38) and bronchial epithelial cells (HBEC3-KT). After 24 h exposure to a Cs-BMP, inflammation was evaluated by immunofluorescent staining for nuclear factor κB (NF-κB) p65 and cyclooxygenase 2 (COX-2). The number of DNA double-strand breaks (DSBs) was also detected by means of phospholylated histone H2AX (γ-H2AX) focus formation assay. Cs-BMP exposure significantly increased NF-κB p65 and COX-2 expressions, which were related to the number of γ-H2AX foci in the cell nuclei. Compared to the uniform (external) exposure to 137Cs γ-rays, NF-κB tended to be more activated in the cells proximal to the Cs-BMP, while both NF-κB p65 and COX-2 were significantly activated in the distal cells. Experiments with chemical inhibitors for NF-κB p65 and COX-2 suggested the involvement of such inflammatory responses both in the reduced radiosensitivity of the cells proximal to Cs-BMP and the enhanced radiosensitivity of the cells distal from Cs-BMP. The data show that local exposure to Cs-BMP leads to biological effects modified by the NF-κB pathway, suggesting that the radiation risk for Cs-BMP exposure can differ from that estimated based on conventional uniform exposure to normal tissues.

Introduction
The incident at the Fukushima Daiichi Nuclear Power Plant (FDNPP) took place after the tsunami disaster in 2011, which emitted large quantities of radioactive materials to the environment [1]. In 2013, insoluble, radioactive cesium-bearing microparticles (Cs-BMPs) were discovered from sites near the FDNPP [2]. Such Cs-BMPs can be classified into two types: Type A with a high specific radioactivity and 1-10 µm diameter, and Type B with a low specific radioactivity and 70-400 µm diameter [3,4]. Cs-BMPs are mainly composed of silicate glass [5,6], which may be formed from glass fibers used in heat insulators covering the pipes [6]. Cs-BMPs have been estimated to be widely dispersed over the greater Kanto region (>250 km away from the FDNPP) [7]. Considering that Cs-BMPs are so small in size and light in weight, Cs-BMPs can reach the human respiratory system. From the viewpoint of radiation protection, it is pivotal to investigate the radiobiological impact of Cs-BMPs.
The radiation risk posed by the intake of internal exposure to radioactive cesium is conventionally estimated from the organ dose given by uniform exposure to soluble cesium. Meanwhile, Cs-BMPs are assumed to adhere in the long term to normal tissues due to their insoluble nature. Considering local energy deposition around a Cs-BMP [8], normal tissues are assumed to be chronically and partially exposed at a microenvironmental scale. However, such heterogeneous dose distribution within the tissue has not been considered in evaluating the radiation risk of Cs-BMPs. Considering such gaps, the conventional risk assessment based on the uniform exposure at an organ scale is insufficient. In addition to the estimation of the intra-tissue dose distribution and biokinetics [9,10], the accumulation of the fundamental in vitro data on local exposure is needed for a better understanding of the radiation risk after exposure to Cs-BMPs.
There are several reports on responses of human normal cells to local radiation exposure [8,11,12]. Among them, our previous in vitro study reported on the spatial distribution of DNA double-strand breaks (DSBs), which was detected by the histone H2AX phosphorylated on serine 139 (γ-H2AX foci) at DSB sites [13,14], after 24 h exposure to Cs-BMP with 505.7 Bq [8]. Our previous results suggest that local exposure to a Cs-BMP leads to an increase in DNA damage to distal cells and a decrease in DNA damage to proximal cells. Therefore, the intercellular signaling between irradiated cells and non-irradiated cells (i.e., via non-targeted effects or bystander effects of radiation [15][16][17][18][19]) may modify radiation responses during local exposure. However, other endpoints have not been evaluated, and the underlying mechanisms remain unclear. Therefore, further studies are necessary for clarifying the underlying mechanisms.
Among various radiobiological endpoints, inflammatory responses have been implicated in carcinogenesis [20][21][22][23][24]. It is well known that the inflammatory responses can be quantified by the signaling pathways involving nuclear factor-kappa B (NF-κB) p65 nuclear translocation and cyclooxygenase-2 (COX-2) expression [25]. NF-κB is also a transcription factor that can bind the kappa immunoglobulin-light chain enhancer [26], which triggers the gene expressions responsible for proliferation and anti-apoptosis. The transcription factor activation induces cytokines responsible for immune reactions (such as tumor necrosis factor α (TNF-α) and interleukins IL-1, IL-6 and IL-8). Due to the adhesion molecules that attract leukocytes to the inflammation sites, it is interpreted that dysregulation of the transcription factor is related to chronic inflammatory diseases and cancer development. Meanwhile, COX-2 is a secondary signaling molecule (downstream of NF-κB [25]) that is produced after stimulation with cytokines and mitogens [27], which is known to be involved in inflammation processes. As inflammatory responses are mediated by NF-κB p65 and COX-2 pathways, the evaluation of the role of these signaling pathways is of importance in discussing radiation effects after inhaling Cs-BMPs. Here, we set out to investigate the inflammatory signaling pathways after Cs-BMP exposure, and evaluate the relationship between inflammatory response and DSB induction, in comparison to responses following uniform exposure to 137 Cs γ-rays.

Irradiation Setups
Cells seeded on the glass-based dish with ϕ35 mm (3911-035, IWAKI, Chiyoda, Japan) were continuously exposed to the Cs-BMP (the sample ID: CF-01 [4]) for 24 h using the microcapillary (MP-020, MicroSupport Co., Ltd., Shizuoka, Tokyo), as described in [8]. In our previous study [8], there was no significant difference between 24 h exposure and 48 h exposure, so we employed 24 h exposure in this study as well. The Cs-BMP used in this study was classified as a Type B particle composed of 94.5% 137 Cs with 455.2 Bq and 5.5% 134 Cs with 26.5 Bq as of 13 January 2020. The irradiation experiments were repeatedly performed at several times during the period from 13 January 2020 to 29 July 2021. Figure 1A illustrates the experimental geometry using the Cs-BMP enclosed in the microcapillary. Figure 1B is the dose profile around the Cs-BMP obtained using the Particle and Heavy Ion Transport code System (PHITS) version 3.08 [28], adapting the electron gamma shower (EGS) mode [29] and RI source database (ICRP07) [30]. Note that the dose profile was evaluated for each irradiation experiment. The cutoff energies for photons and electrons were set as 1 keV. Based on the PHITS calculation, the mean boundary between the β-ray dominant area and γ-ray dominant area was found to be 1650 µm, as described in [8].

Irradiation Setups
Cells seeded on the glass-based dish with φ35 mm (3911-035, IWAKI, Chiyoda, Japan) were continuously exposed to the Cs-BMP (the sample ID: CF-01 [4]) for 24 h using the microcapillary (MP-020, MicroSupport Co., Ltd., Shizuoka, Tokyo), as described in [8]. In our previous study [8], there was no significant difference between 24 h exposure and 48 h exposure, so we employed 24 h exposure in this study as well. The Cs-BMP used in this study was classified as a Type B particle composed of 94.5% 137 Cs with 455.2 Bq and 5.5% 134 Cs with 26.5 Bq as of 13 January 2020. The irradiation experiments were repeatedly performed at several times during the period from 13 January 2020 to 29 July 2021. Figure  1A illustrates the experimental geometry using the Cs-BMP enclosed in the microcapillary. Figure 1B is the dose profile around the Cs-BMP obtained using the Particle and Heavy Ion Transport code System (PHITS) version 3.08 [28], adapting the electron gamma shower (EGS) mode [29] and RI source database (ICRP07) [30]. Note that the dose profile was evaluated for each irradiation experiment. The cutoff energies for photons and electrons were set as 1 keV. Based on the PHITS calculation, the mean boundary between the β-ray dominant area and γ-ray dominant area was found to be 1650 μm, as described in [8].  [28]. The boundary between β-ray dominant area and γ-ray dominant area was found to be 1650 μm, as described in [8].
The homogeneous exposure of the entire area of the cell culture dish (hereafter called uniform exposure) for 24 h to 137 Cs γ-rays was also performed to compare the local exposure to the Cs-BMP. The absorbed dose rates used in this study were 0.450, 0.100, 0.050, 0.010, 0.005 and 0.001 Gy/day. The dose rates were obtained from the measurement using  [28]. The boundary between β-ray dominant area and γ-ray dominant area was found to be 1650 µm, as described in [8].
The homogeneous exposure of the entire area of the cell culture dish (hereafter called uniform exposure) for 24 h to 137 Cs γ-rays was also performed to compare the local exposure to the Cs-BMP. The absorbed dose rates used in this study were 0.450, 0.100, 0.050, 0.010, 0.005 and 0.001 Gy/day. The dose rates were obtained from the measurement using the Farmer-type ionizing radiation chamber (model NE2581, Nuclear Enterprises Ltd., Reading, UK) based on the International Atomic Energy Agency (IAEA) Technical Report Series No. 277 [31] and were validated by the PHITS calculation.
In addition, we also performed a non-uniform exposure to the cell culture dish using 6-MV X-rays from Clinac 600EX, in which the 50% cells were exposed by the placement of cell culture container at the edge of the radiation field. The field size was 10 cm × 10 cm, and the water equivalent depth from the phantom surface was 10 cm. The in-field and out-of-field dose rates were measured using a Farmer-type ionization chamber, which was calibrated according to Japanese Standard Dosimetry 1 [32], and the detailed relative dose profile was obtained by Gafchromic EBT3 film and the PHITS calculation using the phasespace file for Varian Clinac 600C (equivalent to Clinac 6EX) 6MV photons. The geometry and normalized dose profile are depicted in Figure S1, in which the PHITS calculation agreed well with both measured values.
Representative images of the immunofluorescent staining for NF-κB p65 and COX-2 after 24 h exposure to Cs-BMP are shown in Figure 2A, where left, central and right images represent the sham-irradiated group, β-ray dominant area (R ≤ 1650 µm) and γ-ray dominant area (1650 µm < R), respectively. NF-κB and COX-2 positive cells were evaluated by both manual assessment and the fluorescent intensity threshold. In Figure 2A, blue, red and green fluorescent intensities represent the cell nuclei stained by DAPI, NF-κB p65 and COX-2, respectively. Note that yellow fluorescent intensity denotes double-positive cells.

γ-H2AX Focus Formation Assay
Prior to the Cs-BMP exposure, the cells were treated overnight with 1 µM BAY 11-7082 (AG-CR1-0013-M010, AdipoGen, San Diego, CA, USA) as an inhibitor of the NF-κB nuclear translocation, 50 µM NS-398 (70590, Cayman Chemical, Ann Arbor, MI, USA) as the COX-2 inhibitor, or both. As these inhibitors were solved in dimethylsulfoxide (DMSO), we included a mock-treated group in which cells were treated with 0.04% DMSO and exposed to the Cs-BMP for 24 h. After exposure, the cells were fixed by 4% PFA for 10 min on ice, rinsed, and permeabilized in 0.2% Triton X-100 in PBS for 5 min. The cells were then blocked in 1% BSA in PBS for 30 min, and were incubated at 4 • C overnight with primary antibodies against γ-H2AX (ab26350, Abcam, Cambridge, UK) diluted 1:400 by the 1% BSA in PBS. After rinsing with the 1% BSA in PBS three times, the cells were incubated for 2 h with Alexa Fluor 594-conjugated goat-anti-mouse IgG H&L (ab150116, Abcam, Cambridge, UK) diluted by 1:250 by the 1% BSA in PBS. After rinses with the 1% BSA in PBS three times, the cells were incubated with 1 µg/mL DAPI (62248, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min. After rinses with methanol once, γ-H2AX foci were observed under the Keyence BZ-9000 fluorescent microscope.
Representative images of the γ-H2AX foci after 24 h exposure to Cs-BMP are shown in Figure 2B, in which left, central and right images represent the sham-irradiated group, β-ray dominant area (R ≤ 1650 µm) and γ-ray dominant area (1650 µm < R), respectively. The green signal inside the cell nucleus (blue area) represents γ-H2AX focus. To obtain the Cancers 2022, 14, 1045 5 of 15 spatial distribution of nuclear γ-H2AX foci around the Cs-BMP, γ-H2AX focus was evaluated with automated foci count (peak search method) using the ImageJ software [33,34], as described in [8].  Figure 2, left, central and right images represent sham-irradiated group, β-ray dominant area (R < 1650 μm) and γ-ray dominant area (1650 μm < R), respectively. In Figure 2A, blue, red and green fluorescent intensities represent cell nuclei stained by DAPI, NF-κB p65 and COX-2, respectively. Note that yellow fluorescent intensity denotes double-positive cells. In Figure 2B, green signal inside cell nucleus (blue area) represents γ-H2AX focus. Scale bars for Figure 2A and for Figure 2B are 100 and 20 μm, respectively.

γ-H2AX Focus Formation Assay
Prior to the Cs-BMP exposure, the cells were treated overnight with 1 μM BAY 11-7082 (AG-CR1-0013-M010, AdipoGen, San Diego, CA, USA) as an inhibitor of the NF-κB nuclear translocation, 50 μM NS-398 (70590, Cayman Chemical, Ann Arbor, MI, USA) as the COX-2 inhibitor, or both. As these inhibitors were solved in dimethylsulfoxide (DMSO), we included a mock-treated group in which cells were treated with 0.04% DMSO and exposed to the Cs-BMP for 24 h. After exposure, the cells were fixed by 4% PFA for 10 min on ice, rinsed, and permeabilized in 0.2% Triton X-100 in PBS for 5 min. The cells were then blocked in 1% BSA in PBS for 30 min, and were incubated at 4°C overnight with primary antibodies against γ-H2AX (ab26350, Abcam, Cambridge, UK) diluted 1:400 by the 1% BSA in PBS. After rinsing with the 1% BSA in PBS three times, the cells were incubated for 2 h with Alexa Fluor 594-conjugated goat-anti-mouse IgG H&L (ab150116, Abcam, Cambridge, UK) diluted by 1:250 by the 1% BSA in PBS. After rinses with the 1% BSA in PBS three times, the cells were incubated with 1 μg/mL DAPI (62248, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min. After rinses with methanol once, γ-H2AX foci were observed under the Keyence BZ-9000 fluorescent microscope.

Clonogenic Assay
The cultured cells were counted by using a hemocytometer (Erma, Tokyo, Japan) and plated in T25 flasks (156367, Nunc, Waltham, MA, USA). The cells were allowed to adhere overnight prior to irradiation. After exposure to 6-MV-lianc X-rays (Clinac 6EX, Varian, Palo Alto, CA, USA), cells were incubated for 14 days at 37 • C in a humidified atmosphere of 95% air 5% CO 2 . Colonies were fixed with methanol and stained with 2% Giemsa solution (Kanto Chemical Co. Inc., Tokyo, USA). When calculating surviving fraction, the colonies located in the penumbra regions (−1.0 < x [cm] < 1.0 in Figure S1) were excluded. The surviving fraction is the ratio of plating efficiency of the irradiated group to that of the non-irradiated group. The PHITS calculation showed that the out-of-field dose relative to the in-field dose is 5.0% on average ( Figure S1).

Statistics
The significant differences among mean values in inflammatory responses (NF-κB p65 nuclear translocation and COX-2 expression) were evaluated by a multiple comparison method, the Tukey-Kramer test. Meanwhile, because the nuclear γ-H2AX foci did not follow the normal distribution, and there was no homoscedasticity between the shamirradiated group and irradiated group, the significant differences of nuclear foci in number were evaluated by using a multiple comparison method, the Scheffe's F test. Based on the statistical indices, we evaluated the impact of local Cs-BMP exposure on inflammatory signaling and DNA damage responses. To evaluate the impact of non-uniform exposure on clonogenic survival, we used a paired t-test. Based on the p-value, we evaluated the impact of non-uniform exposure on cell survival, compared to the uniform-field exposure.

Spatial Distribution of Inflammation under a Cs-BMP Exposure
We measured the spatial distribution of the inflammatory responses around a Cs-BMP. Figure 3 shows the fractions of the NF-κB-positive, COX-2-positive and double-positive cells after 24 h exposure to the Cs-BMP, in WI-38 cells ( Figure 3A) and HBEC3-KT cells ( Figure 3B). The boundary between the β-ray and γ-ray dominant areas was 1650 µm. Here, we compared four groups, i.e., non-irradiated cells, all exposed cells at a radial distance R ≤ 3300 µm, the proximal cells at R ≤ 1650 µm and the distal cells at 1650 < R ≤ 3300 µm. There was a significant increase in the NF-κB p65 nuclear translocation in both cell lines located within a 3300 µm distance from the Cs-BMP. The dual activation of NF-κB and COX-2 was observed in the proximal region (≤1650 µm) for WI-38 cells and in the entire region for HBEC3-KT cells. The pattern of the dual activation for NF-κB and COX-2 resembles that of nuclear foci of γ-H2AX after the Cs-BMP exposure reported in our previous paper [8].  As shown in Figure 1B, the energy deposited within cells gradually decreases with increasing radial distance from a Cs-BMP. In this experiment, the distance giving 1 mGy, at which a human fibroblast nucleus is traversed on average by approximately one electron track [35], was ~1550 μm. In this regard, the cells at >1550 μm can be categorized as bystander cells (non-hit cells). The bystander effects (intercellular signaling) can induce not only DNA damage [36][37][38] but also inflammatory responses [25,39,40] in non-hit cells. Therefore, the significant dual activation can predominantly be due to the bystander effects. In general, the diffusion distance of intercellular signals is 90-100 μm for calcium [41] and < 800 μm for apoptosis induction [42]. However, the range seems to be much longer in this experimental system.

Comparison of Inflammation between Cs-BMP and 137 Cs γ-rays
The fractions of the NF-κB-positive, COX-2-positive and double-positive cells after the Cs-BMP exposure were next compared to those after the uniform exposure to 137 Cs γrays. Figure 4 shows a comparison between local exposure to the Cs-BMP and uniform exposure to 137 Cs γ-rays, where Figure 4A,B show NF-κB p65 nuclear translocation, Figure  4C,D show COX-2 and Figure 4E,F show dual activation. From the left bar graph in each As shown in Figure 1B, the energy deposited within cells gradually decreases with increasing radial distance from a Cs-BMP. In this experiment, the distance giving 1 mGy, at which a human fibroblast nucleus is traversed on average by approximately one electron track [35], was~1550 µm. In this regard, the cells at >1550 µm can be categorized as bystander cells (non-hit cells). The bystander effects (intercellular signaling) can induce not only DNA damage [36][37][38] but also inflammatory responses [25,39,40] in non-hit cells. Therefore, the significant dual activation can predominantly be due to the bystander effects. In general, the diffusion distance of intercellular signals is 90-100 µm for calcium [41] and < 800 µm for apoptosis induction [42]. However, the range seems to be much longer in this experimental system.

Comparison of Inflammation between Cs-BMP and 137 Cs γ-rays
The fractions of the NF-κB-positive, COX-2-positive and double-positive cells after the Cs-BMP exposure were next compared to those after the uniform exposure to 137 Cs γ-rays. Figure 4 shows a comparison between local exposure to the Cs-BMP and uniform exposure to 137 Cs γ-rays, where Figure 4A,B show NF-κB p65 nuclear translocation, Figure 4C,D show COX-2 and Figure 4E,F show dual activation. From the left bar graph in each panel, there was no difference between sham-irradiated groups for both irradiation regimens in both cell lines. The local exposure to Cs-BMP could induce significant activation of NF-κB p65 in the wide dose range ( Figure 4A,B). In particular, the cells closest to the Cs-BMP tended to show increased translocation of NF-κB p65, compared to those after uniform exposure. Meanwhile, the activation of the COX-2 singling pathway and the double pathways tended to increase in a low-dose range below approximately 0.05 Gy ( Figure 4E,F), which intriguingly is in accordance with the value of γ-H2AX focus formation reported in our previous study [8].  We evaluated the relationship between the increase in double-positive cells and the number of radiation-induced γ-H2AX foci. Figure 5A shows the relation for Cs-BMP exposure and Figure 5B shows that for 137 Cs γ-ray exposure (the data on γ-H2AX foci taken from our previous report [8]). As shown in Figure 5, the inflammatory signaling pathways are related to the nuclear number of γ-H2AX foci (DSBs). Ataxia telangiectasia mutated and the NF-κB modulator cooperate to activate the NF-κB pathway in response to DNA damage [43]. Additionally, the cytokines, such as IL-6 and IL-8, result in the secondary activation of NF-κB and COX-2 pathways, inducing DNA damage in bystander cells [25,44,45]. Thus, a correlation between radiation-induced γ-H2AX foci and an increase in the double-activation seems to be reasonable. However, the R 2 value for Cs-BMP exposure In each panel, the left bar graph shows the levels at 0 Gy. The blue and red symbols (*, **) indicate the 5% and 1% significant difference, respectively, compared to sham-irradiated groups. The error bar means the standard error of the mean (s.e.m.). The black symbols represent significant difference between local exposure to the Cs-BMP and uniform exposure to 137 Cs γ-rays. n.s., non-significant.
We evaluated the relationship between the increase in double-positive cells and the number of radiation-induced γ-H2AX foci. Figure 5A shows the relation for Cs-BMP exposure and Figure 5B shows that for 137 Cs γ-ray exposure (the data on γ-H2AX foci taken from our previous report [8]). As shown in Figure 5, the inflammatory signaling pathways are related to the nuclear number of γ-H2AX foci (DSBs). Ataxia telangiectasia mutated and the NF-κB modulator cooperate to activate the NF-κB pathway in response to DNA damage [43]. Additionally, the cytokines, such as IL-6 and IL-8, result in the secondary activation of NF-κB and COX-2 pathways, inducing DNA damage in bystander cells [25,44,45]. Thus, a correlation between radiation-induced γ-H2AX foci and an increase in the double-activation seems to be reasonable. However, the R 2 value for Cs-BMP exposure is lower than that for uniform exposure (see Figure 5), due to DNA damage responses modified by the NF-κB pathway. Lam et al. reported higher activation of phosphorylated NF-κB when 2.5% of the cell population was irradiated than when 100% was irradiated, which is involved in rescue effect induction (reduction of 53BP1 foci) [46]. Considering these, NF-κB and COX-2 pathways might play an important role in modifying the DNA damage responses under Cs-BMP exposure. However, we separately performed immunofluorescent staining for inflammatory signaling expression and γ-H2AX focus formation assay due to limited function of the fluorescent microscopy used in this study. In future, the triple staining technique is expected to enable a clear correlation between DNA damage and inflammation signaling to be obtained. In future, the triple staining technique is expected to enable a clear correlation between DNA damage and inflammation signaling to be obtained. To discuss the trend using the sufficient amount of experimental data, we used the data on γ-H2AX foci in our previous report [8].

DSB Induction when Inflammatory Signaling Is Inhibited
To further investigate the relationship between inflammatory signaling and DNA damage response, we used 1 μM Bay 11-7082 and 50 μM NS-398 as inhibitors of NF-κB p65 and COX-2 pathways, respectively [39,47]. The effectiveness of these inhibitor concentrations was validated, as shown in Figure S2, showing that NF-κB p65 and COX-2 activations decline to the non-irradiated level for both WI-38 and HBEC3-KT cells. At these concentrations, we measured nuclear γ-H2AX foci as a function of the radial distance from the Cs-BMP for various inhibitor treatments. Figure 6 shows the nuclear γ-H2AX foci after the Cs-BMP exposure for various treatments with inflammatory pathway inhibitors, in which the upper panel ( Figure 6A-C) shows the WI-38 cell line and the lower panel ( Figure 6D-F) shows the HBEC3-KT cell line. The spatial distributions of nuclear γ-H2AX foci for various treatments are summarized in Figures S3 and S4, from which two areas were selected: one is closest to Cs-BMP (i.e., at 0-300 μm), and the other is the boundary between the β-ray dominant and γ-ray dominant areas (1650-1950 μm). The left panel shows the sham-irradiated cells, the middle panel shows the cells located at 0-300 μm from the Cs-BMP and the right panel shows the cells located at 1650-1950 μm from the Cs-BMP. In the sham-irradiated WI-38 cells To discuss the trend using the sufficient amount of experimental data, we used the data on γ-H2AX foci in our previous report [8].

DSB Induction when Inflammatory Signaling Is Inhibited
To further investigate the relationship between inflammatory signaling and DNA damage response, we used 1 µM Bay 11-7082 and 50 µM NS-398 as inhibitors of NF-κB p65 and COX-2 pathways, respectively [39,47]. The effectiveness of these inhibitor concentrations was validated, as shown in Figure S2, showing that NF-κB p65 and COX-2 activations decline to the non-irradiated level for both WI-38 and HBEC3-KT cells. At these concentrations, we measured nuclear γ-H2AX foci as a function of the radial distance from the Cs-BMP for various inhibitor treatments. Figure 6 shows the nuclear γ-H2AX foci after the Cs-BMP exposure for various treatments with inflammatory pathway inhibitors, in which the upper panel ( Figure 6A-C) shows the WI-38 cell line and the lower panel ( Figure 6D-F) shows the HBEC3-KT cell line. The spatial distributions of nuclear γ-H2AX foci for various treatments are summarized in Figures S3 and S4, from which two areas were selected: one is closest to Cs-BMP (i.e., at 0-300 µm), and the other is the boundary between the β-ray dominant and γ-ray dominant areas (1650-1950 µm). The left panel shows the sham-irradiated cells, the middle panel shows the cells located at 0-300 µm from the Cs-BMP and the right panel shows the cells located at 1650-1950 µm from the Cs-BMP. In the sham-irradiated WI-38 cells (Figure 6A), there were no significant difference among the sham-irradiated groups, indicating the lack of impact of these inhibitors on DNA damage induction for WI-38 cells. Meanwhile, these inhibitors could affect the number of γ-H2AX foci in sham-irradiated HBEC3-KT cells ( Figure 6B). From these results, we calculated the γ-H2AX foci relative to the shamirradiated group, and evaluated the impact of the inhibition of NF-κB and COX-2 on DNA damage induction after Cs-BMP exposure. Figure 6F shows that the inhibition of NF-κB and COX-2 reduced DNA damage (DSB) induction in the HBEC3-KT cells at 1650-1950 µm, suggesting that DNA damage induction under low-dose-rate exposure is involved in the activation of the inflammatory signaling pathway by bystander effects [44,48,49]. In the cells at 0-300 µm, the COX-2 suppression also reduced DSB induction ( Figure 6B,E); however, the NF-κB p65 suppression elicited the opposite effects (enhancing DSB induction). In support of this, NF-κB inhibition induced detrimental effects on the 53BP1 foci increase [50] and differentiation (culture phenotype) [51]. involved in the activation of the inflammatory signaling pathway by bystander effects [44,48,49]. In the cells at 0-300 μm, the COX-2 suppression also reduced DSB induction ( Figure 6B,E); however, the NF-κB p65 suppression elicited the opposite effects (enhancing DSB induction). In support of this, NF-κB inhibition induced detrimental effects on the 53BP1 foci increase [50] and differentiation (culture phenotype) [51]. NF-κB inhibition downregulates COX-2 expression [25,51], thereby mitigating inflammation ( Figure 6C,F). In particular, the subsequent COX-2 expression positively modulates bystander cells [44]. Additionally, the in vitro study using aminoguanidine showed that inducible nitric oxide synthase (iNOS) plays a similar role in inducing bystander effects on DSB induction [38]. Reactive oxygen species (ROS) was identified as the messenger of DNA damage induction by bystander effects [8]. Meanwhile, the NF-κB pathway not only stimulates DSB repair, particularly homologous recombination (HR) [52][53][54], but also is associated with a decrease in the ROS level in irradiated cells [55,56]. The NF-κB signaling pathway has been attributed to an inducer for radioresistance due to its antiapoptotic function [57]. Our previous report suggested that γ-H2AX focus formation was less manifested in the cells proximal to the Cs-BMP, which was interpreted by the reduced yield of early damage (so called protective effects) [8]. Nitric oxide-mediated bystander effects can be a trigger to cause radioresistance [58]; however, the inhibition of iNOS did not diminish protective effects [59,60]. Therefore, it is suggested that the upregulation by NK-κB plays a key role in the protective effects. The symbols (*, **) indicate the 5% and 1% significant difference, respectively, while the value represents the p-value. n.s., non-significant. The symbol on each bar graph indicates the significance or non-significance compared to sham-irradiated group.  (s.d.). The symbols (*, **) indicate the 5% and 1% significant difference, respectively, while the value represents the p-value. n.s., non-significant. The symbol on each bar graph indicates the significance or non-significance compared to sham-irradiated group. NF-κB inhibition downregulates COX-2 expression [25,51], thereby mitigating inflammation ( Figure 6C,F). In particular, the subsequent COX-2 expression positively modulates bystander cells [44]. Additionally, the in vitro study using aminoguanidine showed that inducible nitric oxide synthase (iNOS) plays a similar role in inducing bystander effects on DSB induction [38]. Reactive oxygen species (ROS) was identified as the messenger of DNA damage induction by bystander effects [8]. Meanwhile, the NF-κB pathway not only stimulates DSB repair, particularly homologous recombination (HR) [52][53][54], but also is associated with a decrease in the ROS level in irradiated cells [55,56]. The NF-κB signaling pathway has been attributed to an inducer for radioresistance due to its antiapoptotic function [57]. Our previous report suggested that γ-H2AX focus formation was less manifested in the cells proximal to the Cs-BMP, which was interpreted by the reduced yield of early damage (so called protective effects) [8]. Nitric oxide-mediated bystander effects can be a trigger to cause radioresistance [58]; however, the inhibition of iNOS did not diminish protective effects [59,60]. Therefore, it is suggested that the upregulation by NK-κB plays a key role in the protective effects.

Cell Survival after Heterogeneous Exposure to X-rays
The NF-κB pathway also negatively controls apoptosis, i.e., towards cell survival [25,57]. To further discuss the normal tissue effects of heterogeneous exposure, the surviving fractions of WI-38 cells and HBEC3-KT cells were measured using the non-uniform exposure technique [59,60]. Figure 7 shows the surviving fraction measured by a clonogenic assay, where the green symbol shows the survival after uniform exposure, and the blue and red symbols show the survival of in-field cells and out-of-field cells, respectively. As shown in Figure 7, the in-field cells under the non-uniform exposure showed lower radiosensitivity than those under uniform-field exposure (indicating protective effects), which showed a tendency similar to the observation in AG01522 normal human foreskin fibroblasts [59]. The HBEC3-KT cells exhibited more protective effects than the WI-38 cells. Meanwhile, the out-of-field cells under the non-uniform exposure showed higher radiosensitivity than those under the uniform-field exposure at the same absorbed dose. Note that on average, the out-of-field dose was 5% of the in-field dose. In the same manner as for the protective effects, HBEC3-KT cells were radiosensitive to intercellular signaling, leading to more cell killing compared to WI-38 cells.
The out-of-field cells exhibited enhanced radiosensitivity due to intercellular signaling from in-field cells to out-of-field cells, such as via NO and ROS [59,60]. Several reports have shown that the inhibition of iNOS (an upstream of NO) by aminoguanidinecan and inhibition of ROS by DMSO can rescue the enhanced radiosensitivity of out-of-field cells [37,38,61]. We also detected the significant activation of NF-κB and COX-2 in out-of-field cells at 2 h after non-uniform exposure to 4 Gy ( Figure S5A) as bystander responses [25,44,48]. The persistent COX-2 expression was also detected at 16 h after exposure ( Figure S5B). Therefore, the death of out-of-field cells is attributed to inflammatory responses via intercellular signaling. By changing the irradiation area of culture flask, i.e., 25%, 50%, 75% and 100%, we also measured in-field and out-of-field cell survival ( Figure S6A). The bystander effects in out-of-field cells seemed to be saturated for a larger field size than 50%. Meanwhile, the protective effects were maximal at the smallest in-field size of 25% ( Figure S6B). Ojima et al. reported the increase in nuclear 53BP1 foci with X-irradiated field size [12], and Lam et al. reported rescue effects on 53BP1 foci at 12 h post-irradiation with a 2.5% irradiated cell population [46]. The tendency of survival data ( Figure S6) agrees well with these data on 53BP1 foci [12,46]. The potential underlying mechanisms of protective effects might be attributable to the reduction in early DNA damage by antioxidants [8,59] or stimulated DNA repair [52][53][54]. The positive effects for irradiated cells might result from NF-κB/Rel and inhibitor of NF-κB (IκB) gene families as mediators of immune responses [44,62] by intercellular communication from irradiated to non-irradiated cells and vice versa (so called bystander cross-talk) [63]. However, due to the limited amount of experimental data, the underlying mechanisms on inducing protective effects remain uncertain, warranting further experiments.
blue and red symbols show the survival of in-field cells and out-of-field cells, respectively. As shown in Figure 7, the in-field cells under the non-uniform exposure showed lower radiosensitivity than those under uniform-field exposure (indicating protective effects), which showed a tendency similar to the observation in AG01522 normal human foreskin fibroblasts [59]. The HBEC3-KT cells exhibited more protective effects than the WI-38 cells. Meanwhile, the out-of-field cells under the non-uniform exposure showed higher radiosensitivity than those under the uniform-field exposure at the same absorbed dose. Note that on average, the out-of-field dose was 5% of the in-field dose. In the same manner as for the protective effects, HBEC3-KT cells were radiosensitive to intercellular signaling, leading to more cell killing compared to WI-38 cells. Red, out-of-field cells. There were significant differences between in-field survival after non-uniform exposure and that after uniform-field exposure, except for 1 Gy and 2 Gy in WI-38 cells. The inset represents the low-dose range. The out-of-field dose is 5%, on average, of in-field dose.
The out-of-field cells exhibited enhanced radiosensitivity due to intercellular signaling from in-field cells to out-of-field cells, such as via NO and ROS [59,60]. Several reports have shown that the inhibition of iNOS (an upstream of NO) by aminoguanidinecan and inhibition of ROS by DMSO can rescue the enhanced radiosensitivity of out-of-field cells [37,38,61]. We also detected the significant activation of NF-κB and COX-2 in out-of-field cells at 2 h after non-uniform exposure to 4 Gy ( Figure S5A) as bystander responses [25,44,48]. The persistent COX-2 expression was also detected at 16 h after exposure (Figure S5B). Therefore, the death of out-of-field cells is attributed to inflammatory responses via intercellular signaling. By changing the irradiation area of culture flask, i.e., 25%, 50%, 75% and 100%, we also measured in-field and out-of-field cell survival ( Figure S6A). The bystander effects in out-of-field cells seemed to be saturated for a larger field size than 50%. Meanwhile, the protective effects were maximal at the smallest in-field size of 25% ( Figure S6B). Ojima et al. reported the increase in nuclear 53BP1 foci with X-irradiated field size [12], and Lam et al. reported rescue effects on 53BP1 foci at 12 h post-irradiation Red, out-of-field cells. There were significant differences between infield survival after non-uniform exposure and that after uniform-field exposure, except for 1 Gy and 2 Gy in WI-38 cells. The inset represents the low-dose range. The out-of-field dose is 5%, on average, of in-field dose.

A Scenario Model of the Signaling Pathways under Cs-BMP Exposure
Based on our in vitro experimental results, we modeled scenarios on signaling pathways regulating radiobiological effects modulated by intercellular signaling under Cs-BMP exposure. Figure 8 shows the relationship among inflammatory signaling responses, DSB induction and cell death during exposure to Cs-BMP (non-uniform exposure). Figure 8A is the schematic illustration of Cs-BMP exposure and dose of Cs-BMP. Figure 8B is a hypothetical model of the signaling pathways regulating radiobiological effects under Cs-BMP exposure.
The energy locally deposited in proximity to a Cs-BMP, and the boundary between the β-ray dominant area and γ-ray dominant area, was 1650 µm on average ( Figure 1B). During continuous local exposure to Cs-BMP, β-rays and secondary electrons induced DNA damage by energy deposition [64] in mainly proximal cells by targeted effects to directly irradiated cells. Such DNA damage can be repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) [65]. DNA damage by targeted effects activated the NF-κB pathway and downstream COX-2 expression, while intercellular signals induced non-targeted effects and also activated the inflammatory signaling pathway (NF-κB and COX-2) (see Figures 3 and 4), as well as iNOS expression [59,60]. Considering ROS-mediated DNA damage induction in non-hit cells in our previous study [8], the inflammatory signaling finally generated ROS and NO in non-hit cells (distal cells) and induced DNA damage through bystander effects [8,59], leading to enhanced cell death induction (see Figure 7) [59]. Meanwhile, the NF-κB pathway suppressed the ROS level using antioxidants and reduced DNA damage induction (see Figure 6B,E) [46,55,56,59] (DNA repair stimuli [52][53][54]), and controlled antiapoptic function in irradiated cells (Figure 7) [57,59]. Altogether, intercellular signaling induced protective effects in irradiated (hit) cells and bystander effects in non-irradiated (non-hit) cells, respectively. Cancers 2022, 14, x FOR PEER REVIEW 13 of 16

Conclusions
Assuming that Cs-BMP adheres in the long term to the lung, we investigated the relationship between the inflammatory signaling and DNA damage responses during continuous local exposure to a Cs-BMP (mimicking localized internal exposure). We observed the significant activations of NF-κB p65 and COX-2 after 24 h exposure to Cs-BMP. Such inflammatory signaling pathways were related to DNA damage induction (evaluated as γ-H2AX focus formation). Compared with responses to uniform exposure to 137 Cs γ-rays (mimicking external exposure), we found that NF-κB signaling plays an important role in protective effects on the irradiated (hit) cells proximal to Cs-BMP and the bystander effects on the non-irradiated (non-hit) cells distal from Cs-BMP. This work shows that the local exposure to Cs-BMP induces different biological effects modified by inflammatory signaling responses from the conventional effects of uniform exposure.
The radiation risk posed by internal exposure due to the intake of radioactive cesium is conventionally estimated based on uniform exposure to soluble cesium at an organ scale. The present in vitro data suggest that the risk assessment based on uniform exposure may not apply for continuous local exposure to Cs-BMP. However, the scientific data reporting on radiobiological impacts under non-uniform exposure are limited. In particular, in vitro experiments focusing on mutation frequency (or direct observation of

Conclusions
Assuming that Cs-BMP adheres in the long term to the lung, we investigated the relationship between the inflammatory signaling and DNA damage responses during continuous local exposure to a Cs-BMP (mimicking localized internal exposure). We observed the significant activations of NF-κB p65 and COX-2 after 24 h exposure to Cs-BMP. Such inflammatory signaling pathways were related to DNA damage induction (evaluated as γ-H2AX focus formation). Compared with responses to uniform exposure to 137 Cs γ-rays (mimicking external exposure), we found that NF-κB signaling plays an important role in protective effects on the irradiated (hit) cells proximal to Cs-BMP and the bystander effects on the non-irradiated (non-hit) cells distal from Cs-BMP. This work shows that the local exposure to Cs-BMP induces different biological effects modified by inflammatory signaling responses from the conventional effects of uniform exposure.
The radiation risk posed by internal exposure due to the intake of radioactive cesium is conventionally estimated based on uniform exposure to soluble cesium at an organ scale. The present in vitro data suggest that the risk assessment based on uniform exposure may not apply for continuous local exposure to Cs-BMP. However, the scientific data reporting on radiobiological impacts under non-uniform exposure are limited. In particular, in vitro experiments focusing on mutation frequency (or direct observation of carcino-genesis) or using 3D culture models, as well as in vivo studies, are essential for a more precise understanding of radiobiological impacts under heterogeneous exposure. Additionally, mechanistic studies using biophysical models will offer an effective approach to mechanistically interpreting the scenario of intercellular communication under continuous heterogeneous exposure.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14041045/s1, Figure S1: dose profile of non-uniform exposure to 6MV-linac X-rays; Figure S2: Test for evaluating the effectiveness of the inhibitor; Figures S3 and S4: Spatial distribution of nuclear γ-H2AX foci for various inhibitor treatments for WI-38 cell line and HBEC3-KT cell line, respectively; Figure S5: NF-κB and COX-2 levels after non-uniform exposure; Figure S6: Dependency of irradiation area size on in-and out-of-field cell survival.


Astrocytes and Inflammatory T Helper Cells: A Dangerous Liaison in Multiple Sclerosis

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder of the central nervous system (CNS) characterized by the recruitment of self-reactive T lymphocytes, mainly inflammatory T helper (Th) cell subsets. Once recruited within the CNS, inflammatory Th cells produce several inflammatory cytokines and chemokines that activate resident glial cells, thus contributing to the breakdown of blood-brain barrier (BBB), demyelination and axonal loss. Astrocytes are recognized as key players of MS immunopathology, which respond to Th cell-defining cytokines by acquiring a reactive phenotype that amplify neuroinflammation into the CNS and contribute to MS progression. In this review, we summarize current knowledge of the astrocytic changes and behaviour in both MS and experimental autoimmune encephalomyelitis (EAE), and the contribution of pathogenic Th1, Th17 and Th1-like Th17 cell subsets, and CD8+ T cells to the morphological and functional modifications occurring in astrocytes and their pathological outcomes.

INTRODUCTION
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) affecting more than 2.5 million people worldwide, with a 2020 global prevalence of 35.9 per 100000 people (1). In the majority of patients, MS begins with a single clinically isolated syndrome (CIS) of neurological dysfunction that resolves over time. After the initial CIS, most patients have a second relapse and develop the relapsing-remitting MS (RRMS) form (2). Relapses are characterized by CNS inflammation and confluent area of demyelination in the white and grey matter of the brain and spinal cord caused by the loss of oligodendrocytes and myelin sheaths (3). One to two decades post-diagnosis, 15-30% of RRMS patients develop secondary progressive MS (SPMS) that is characterised by gradual neuroaxonal loss and brain atrophy, thus leading to patient disability and neurodegeneration (4). About 15% of patients develop an irreversible primary progressive form (PPMS) from the onset characterized by chronic demyelinated lesions in the white matter, axonal loss, diffuse and focal demyelination of the grey matter and neurodegeneration (5).
Despite the exact causes of MS remain still unknown, the disease is known to arise in genetically susceptible individuals (6, 7) by a complex interplay between environmental factors (8) and dysregulated immune responses (9). Several studies performed in murine models of experimental autoimmune encephalomyelitis (EAE) to explain MS pathophysiology, validated the hypothesis that MS is an autoimmune disorder characterized by the infiltration within the CNS of adaptive self-reactive immune cells, which cause demyelination and remyelination events, thus leading to the loss of sensory and motor functions (10). It is still an open question whether the initial MS triggering insult occurs within the CNS (intrinsic model), presumably affecting the oligodendrocytes and favouring the release of CNS antigens to the periphery, or whether it takes place outside the CNS (extrinsic model) leading to the activation of aberrant adaptive immune responses targeting CNS antigens (9). Independently of the place where the triggering events occur, peripheral innate and adaptive immune cells, especially autoreactive inflammatory T helper (Th) cells, cross the blood-brain barrier (BBB) and release inflammatory mediators in the brain that affect the function of resident glial cells, leading to astrogliosis, oligodendrocyte loss and axonal degeneration (11,12).
Astrocytes are star-shaped glial cells that play a pivotal role in maintaining CNS homeostasis (13). Through highly ramified processes, astrocytes contact several cells within the CNS contributing to the formation, activity and plasticity of neuronal synapses (14), providing neurotrophic factors and metabolic support to neurons and oligodendrocytes (15)(16)(17)(18) and ensuring the formation and maintenance of BBB integrity (19). At a resting state and in different brain regions, astrocytes are highly heterogeneous in their morphology and functional properties (20). Protoplasmic astrocytes, mainly located in the grey matter, at the hippocampus and cerebral cortex, are characterised by extremely ramified cell bodies, thus allowing them to contact synapses and perform neuromodulation. Fibrous astrocytes, mainly located in the white matter, are smaller with longer and narrower protrusions, which interact with axons at the level of the nodes of Ranvier (21). Besides these two main astrocyte subpopulations, nine more distinct astrocyte-like subtypes have been described on the basis of their morphological features including radial, marginal and perivascular glia located in the cortex of human brain (20). Moreover, the advent of single-cell RNA sequencing (scRNAseq) and single-cell spatial transcriptomics evidenced further diversity and specialization of astrocytes depending on their differential brain localization (22)(23)(24).
In this review, we describe the main astrocytic changes occurring in MS and the role of the crosstalk between inflammatory T cells and astrocytes in amplifying CNS inflammation and MS progression ( Figure 1).

ASTROCYTE DYSREGULATION IN MS
In both MS and EAE, the activation of astrocytes occurs at an early stage and persists into the acute and chronic stages of the disease. Several changes in both morphology and spatial localization of astrocytes have been observed in different stages of the disease (28). Studies in acute EAE showed the presence of hypertrophic reactive astrocytes at a very early stage of the symptomatic phase, even before immune cells cross the BBB and enter into the CNS parenchyma (35,36). In acute MS lesions from post-mortem brain biopsies, hypertrophic reactive astrocytes with damaged end-feet processes were detected in active plaques as well as in the adjacent normal white and grey matter, thus suggesting their pivotal role in both the development and sustainment of the lesions (28,37,38). Astrocytes with a very swollen cytoplasm due to the accumulation of GFAP + filaments were also found in activeacute lesions (28). Furthermore, the phenotypic characterization of reactive astrocytes in active demyelinating MS lesions evidenced that they lose most of their homeostatic functions and acquire a highly inflammatory and neurotoxic phenotype, thus inducing the death of both neurons and mature oligodendrocytes (39,40). As recently evidenced by magnetic resonance imaging (MRI)-informed scRNAseq, astrocytes still remain active in chronic active demyelinated lesions and form an astroglial scar as soon as chronic lesions became inactive (41). Consistently, scRNAseq analysis of CNS samples from EAE mice identified a dominant cluster of high proinflammatory and neurotoxic astrocytes, characterized by increased GM-CSF signalling, NF-kB activation and iNOS expression. The presence of this reactive astrocyte subpopulation was also confirmed in post-mortem brain tissues from MS patients who underwent euthanasia followed by rapid autopsy (42).

BBB Breakdown and Leukocyte Recruitment
The breakdown of the BBB is one crucial hallmark of MS (43,44) that precedes the infiltration of peripheral leukocytes and autoreactive T lymphocytes that, once entered into the CNS, contribute to the development and expansion of MS lesions by damaging various cellular components of the BBB (45)(46)(47)(48)(49)(50). The BBB is a continuous endothelial barrier between the CNS and peripheral blood that provides oxygen and critical nutrients to the CNS and limits the entry of toxic substances and immune cells. The integrity and functionality of the BBB are ensured by the physiological properties of highly specialized endothelial cells (EC) that, by interacting with pericytes, perivascular astrocytes and neurons, form a neurovascular unit (NVU) that limits both paracellular and transcellular movement of cells and solutes (51,52). Perivascular astrocytes are crucial structural and functional components of the BBB that through their end-feet interact with ECs and ensheathe the brain vasculature (53). Disruption of the astroglia/NVU communication has been linked to BBB breakdown in both EAE and MS (37,54). At the NVU, astrocytes produce several vasoactive molecules such as nitric oxide (NO), prostaglandins and arachidonic acid that regulate cerebral blood flow (55). Moreover, the specialized water-channel aquaporin-4 (AQP-4), the inward rectifying potassium channel (Kir) Kir4.1 and the gap junction-forming protein connexin 43 (Cx43) expressed by the end-feet at the level of glia limitans confer to astrocytes the ability to regulate the exchange of water and ions across the BBB (56). Extensive loss of Cx43 with concomitant patchy loss of AQP-4 was observed in actively demyelinating and chronic active lesions of progressive MS patients with high relapse rates (57), thus contributing to the weakened of BBB and to the vasogenic oedema due to increased hydrostatic vascular pressure and shear stress ( Figure 1) (58).
The integrity of the BBB is also maintained by the tight junctions (TJs), large multiple transmembrane proteins containing occludins, claudins and junctional adhesion molecules (JAMs), which mediate tight adhesion between adjacent ECs. Claudins are the major components of BBB TJs and are essential for the maintenance of BBB integrity (19). The downregulation of claudin-5 and claudin-11 at the BBB has been associated with the impairment of barrier function (59,60). In both MS and EAE lesions, reactive astrocytes upregulate thymidine phosphorylase (TYMP) and vascular endothelial growth factor A (VEGFA), which mediate the downregulation of occludin and claudin-5, thus contributing to BBB breakdown (61,62). Furthermore, reactive astrocytes also produce CCchemokine ligand 2 (CCL2), which contributes to the disassembly of TJs (63) and to the downregulation of both occludin and claudin-5 (64).
In addition to produce soluble factors that increase the permeability of the BBB, reactive astrocytes secrete several chemokines that favour the recruitment of circulating leukocytes into the CNS ( Figure 1). CCL2 upregulation was observed in astrocytes from the white matter lesions of both MS post-mortem brains and EAE mice, where it plays a critical role in both macrophage and T cell infiltration into the white matter of spinal cord (31,65,66). In EAE and MS lesions, reactive astrocytes have been also identified as the major source of CCL20 (32,33,67), a chemokine that mediates the recruitment of pathogenic CCR6 + Th17 and Th1-like Th17 cells into the inflamed CNS (68). Furthermore, high levels of CXCL10 production by astrocytes correlated with the accumulation of CXCR3 + Th1 and Th1-like Th17 cells into the inflamed spinal cord and demyelinated lesions in EAE (25) (Figure 1).

Impaired Astrocyte-Neuron Communication in MS
In the CNS, astrocytes are closely associated with neurons, by tightly enwrapping neuronal cell bodies, axons and synapses ( Figure 1). The association between astrocytes and synapses is important to maintain the brain homeostasis and to regulate neuronal synaptic transmission (69). Astrocytes regulate synaptic functions by tuning glutamate concentration in the synaptic cleft (70). Glutamate is the major excitatory neurotransmitter in CNS that, if accumulates in the synaptic and extra-synaptic space, may lead to the hyperexcitation of neurons and neuronal death through a process known as glutamate excitotoxicity (71). After release from presynaptic neurons, glutamate is taken up from post-synaptic receptors such as mGluRs (metabotropic glutamate receptors), NMDARs (N-methyl-D-aspartate receptors) and AMPARs (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors), which in turn transmit the excitatory impulse (72). Excessive and prolonged stimulation of glutamate receptors leads to the depolarization of postsynaptic membranes and mitochondrial Ca 2+ overload that triggers excessive production of reactive oxygen species (ROS) and nitric oxide (NO) and the opening of the mitochondrial transition pore, thus favouring the release of pro-apoptotic proteins and neuronal cell death (73). Astrocytes play a crucial role in removing the excess of glutamate by using specific glutamate transporters such as glutamateaspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), which uptake more than 80% glutamate released in the synaptic cleft. In astrocytes, glutamate is then metabolized by glutamine synthase (GS) into glutamine that in turn is released, taken up by neurons and used for the synthesis of glutamate and gamma-aminobutyric acid (GABA) (71). Increased levels of glutamate were observed in the CSF of RRMS patients with active lesions during relapse as well as in SPMS patients (74). Moreover, in both MS and EAE, impaired glutamate homeostasis has been related to both decreased glutamine synthetase and increased glutaminase, the enzyme responsible for glutamate synthesis, as well as to the downregulation of the glutamate transporters GLT-1 and GLAST (75)(76)(77)(78)(79). In addition to impaired glutamate uptake, reactive astrocytes were also described to release glutamate at the peak stage of EAE by upregulating the glutamate carboxypeptidase II, a metalloprotease that converts the neuropeptide N-acetylaspartylglutamate into Nacetyl aspartate and glutamate (80).
Astrocyte end-feet express several channels and ion transporters, which regulate ion homeostasis and neuronal excitability. In particular, Kir4.1 K + channel, coexpressed together with AQP4 in the astrocytes end-feet, faces neuronal synapses and allows a rapid clearance of K + ions from the extracellular space, thus facilitating the repolarization of neuronal membranes and neuronal firing (81). Reduced levels of Kir4.1 were observed in perivascular astrocytes in both acute and chronic active demyelinated MS lesions (82). The reduction of astroglial Kir4.1 channel was associated to increased serum levels of complement-fixing IgG subclasses, suggesting a role of anti-Kir4.1 autoantibody in amplifying inflammation and tissue damage in MS (82,83).
Another important function of astrocytes is to regulate the brain energy supply required for the transmission of synaptic impulses and neuron functions (18). Astrocytic end-feet rapidly uptake glucose from the brain capillaries and metabolize it in the glycolytic route to generate lactate. Lactate is then transferred through monocarboxylate transporters (MCT) to neurons where it is converted to pyruvate and oxidized for energy production in mitochondria (84). Interestingly, a significant reduction in the expression of genes encoding for both the astrocyte-neuro lactate shuttle (ANLS), including MCT1, and the glutamate-glutamine cycle (GGC), including glutamine synthase and GLT-1, has been observed in the grey matter from post-mortem brain tissues of chronic SPMS and PPMS patients (85). The downregulation of both ANLS and GGC genes observed in MS was also associated with the simultaneous up-regulation of inflammatory cytokines suggesting a role of immune-related signalling in the impairment of astrocyte metabolic functions (85). Consistently, Ponath et al. found that astrocytes derived from pluripotent stem cells of MS patients carrying the risk allele variant rs7665090 G produced large amounts of proinflammatory factors and displayed a significant reduced ability to release lactate and reuptake glutamate after stimulation (86). The further analysis of reactive astrocytes in white matter lesions from post-mortem tissues suggested the presence of harmful hypertrophic astrocytes in MS patients carrying the risk allele variant (86). The impaired metabolic functions of astrocytes observed in MS also involve the synthesis of cholesterol that is required for myelin sheath formation, the maintenance of axonal membrane and synapses integrity (87). Astrocytes from spinal cord, cerebellum and optic nerve of chronic EAE showed a reduction in the expression of several genes involved in cholesterol synthesis. Similar results were observed by the gene expression analyses of astrocytes from optic chiasm autopsy tissues from MS patients (88).
In order to support the correct neuronal effector responses, astrocytes also secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotropic factors (BDNF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF), which are required for the optimal survival, growth and differentiation of neurons and for preventing neurodegeneration (89). Impaired production of neurotrophic factors was observed in astrocytes exposed to T cell-derived inflammatory cytokines in EAE (90). Moreover, in both EAE and chronic MS lesions, astrocytes up-regulated the BDNF receptor TrkB that upon stimulation with BDNF induced a strong release of NO, thus contributing to oxidative stress and neuronal damage (91).

Impaired Astrocyte-Oligodendrocyte Communication in MS
Reactive and hypertrophic astrocytes, accumulating in MS demyelinating lesions, also contribute to oligodendrocyte loss and demyelination by favouring lesion development and progression (38,92). In healthy brains, astrocytes regulate the homeostasis of myelin sheaths by releasing several neurotrophic factors that promote the proliferation of oligodendrocyte progenitor cells (OPCs) their migration and differentiation to oligodendrocytes (93). The platelet-derived growth factor (PDGF) and BDNF secreted by astrocytes promote OPCs proliferation, migration and maturation to myelinating oligodendrocytes (94). During neuroinflammation, astrocytes also exert neuroprotective effects on oligodendrocytes by producing CXCL1 and CNTF, which favour OPCs recruitment to axons and their differentiation into mature myelinating oligodendrocytes, respectively (95). Consistently, reactive astrocytes in acute MS lesions secrete several remyelinating factors (96) and increased BDNF release by reactive astrocytes was found to induce remyelination in a cuprizone-induced demyelination model (97). However, with the progression of MS to a chronic stage, reactive astrocytes form a dense glial scar around the axons and secrete hyaluronan and proteoglycans, thus preventing OPCs recruitment and maturation into the demyelinated areas (97,98). Moreover, astrocytes also supply lipids, especially cholesterol, to oligodendrocytes necessary for myelin synthesis (16,99). This astrocyte-oligodendrocyte network is finely regulated by a physical interaction through connexins Cx30/Cx32 and Cx43/Cx47, which are fundamental for the exchange of potassium ions and metabolic factors required for myelin maintenance (16,100,101). A strong reduction of Cx47 in both cell bodies and proximal oligodendrocyte processes as well as of the astrocyte binding partner Cx43 was observed in EAE lesions. A concomitant loss of Cx32 was also detected within and around the lesions that persisted throughout the disease course (102). Similar results were obtained by immunohistochemical analysis of post-mortem brain tissues from MS patients, where a strong reduction of both oligodendrocyte Cx32 and Cx47 was observed in and around chronic lesions as well as in the normal-appearing white matter (NAWM) (103). On the contrary, the expression of Cx30 and Cx43 on astrocytes was increased in both lesions and NAWM and correlated with astrogliosis and the acquisition by astrocytes of an inflammatory phenotype, while higher Cx32 expression was associated with a longer disease duration (104). So, the loss of connection between oligodendrocytes and reactive astrocytes during chronic inflammation may accelerate MS progression by contributing to demyelination and axonal damage. Consistently, Cx43 loss was associated with a rapidly progressive MS course, oligodendrogliopathy and active demyelinating lesions (57).

DYSREGULATION OF ASTROCYTE FUNCTIONS BY INFLAMMATORY T CELLS IN MS
The immunopathogenesis of MS relies on the recruitment of specific autoreactive Th cell subsets and CD8 + T cells within CNS where they are reactivated and secrete cytokines and chemokines that modulate the activity of several glial cells, including astrocytes (11,105). Among Th cells, Th1, Th17 and Th1-like Th17 cells have been identified as key players of MS pathogenesis, by producing one or more lineage-defining cytokines, which affect several astrocyte functions as discussed below (Figure 1).

Th1-Mediated Regulation of Astrocytes in MS
Th1 cells are a subset of CD4 + T lymphocytes characterized by the expression of the CXC chemokine receptor type 3 (CXCR3), interleukin (IL)-12 receptor (IL-12R) chains b1/b2, the master transcription factor T-bet and by the production of the lineagesignature cytokine IFN-g together with GM-CSF and TNF-a (106). The neuropathological functions of Th1 cells in MS have been extensively studied in EAE animal models (11) and associated to their ability to trigger the activation of resident microglia and their differentiation into a high inflammatory and neurotoxic phenotype (107,108). More recent studies evidenced that Th1 cells and their effector cytokines may also affect the phenotype and functions of astrocytes in MS (109). Human astrocytes, indeed, were found to express IFN-g receptor (IFNGR) that was also up-regulated in the cortex of postmortem MS brain tissues and associated with the acquisition of a neurotoxic phenotype (110). Silencing of IFN-g signalling in murine astrocytes, suppressed EAE by inhibiting inflammatory chemokine production and the infiltration of Th1 and Th17 cells into the CNS (111,112). Moreover, IFN-g-treated astrocytes upregulated the expression of chemokines such as CCL20, CXCL10 and CXCL12 involved in the recruitment of both Th1 and Th17 cells into the CNS, and CCL2 (90,113) that contributes to BBB breakdown by inducing both disassembly and downregulation of TJs (63,64). In addition to favour the infiltration of inflammatory T cells within CNS, IFN-g-activated astrocytes were also described to promote the proliferation of myelinspecific T cells during EAE by up-regulating major histocompatibility complex class II (MHC-II) molecules and contributing to the reactivation of pathogenic T lymphocytes as antigen-presenting cells (APC) (114). Interestingly, astrocytes in chronic active lesions from post-mortem MS brain tissues were found to express MHC-II together with B7.1 and B7.2 (115,116), two important costimulatory molecules that are required for optimal APC functions and up-regulated by IFN-g (117,118). However, IFN-g was also described to mediate protective effects on astrocytes during chronic EAE. Smith et al. observed an exacerbation of chronic EAE, an increase of the lesion size and enhanced oxidative stress, in mice with IFNGR-deficient astrocytes (119). Similar results were obtained in EAE transgenic mice expressing a signalling deficient dominant negative IFNGR1 on astrocytes (120).
Th1-derived cytokines have been also implicated in polarizing astrocytes to a neurotoxic phenotype. In astrocytes from EAE mice, Th1-derived cytokines such as IFN-g and GM-CSF impaired the expression of neurotrophic factors such as NGF, CNTF and BDNF, and up-regulated the expression of NO synthase (90). Moreover, the loss of Cx43 in astrocytes from acute demyelinating MS lesions has been recently associated to Th1-derived IFN-g via microglia-dependent production of IL-1b, thus contributing to the disruption of astrocyte intercellular communications and MS progression (121).

Th17-Mediated Regulation of Astrocytes in MS
Th17 cells are characterized by the expression of CCR6, CCR4, CD161, IL-23R, IL-1R, the master transcription factor retinoic acid receptor-related orphan nuclear receptor gt (RORgt) and the production of the lineage-signature cytokines IL-17A-F and IL-21 together with IL-22 (122). The pathogenic functions of Th17 cells in MS have been associated to BBB breakdown and CNS inflammation (123,124) by targeting both resident microglia and astrocytes (90,107). Astrocytes, indeed, express a functional IL-17R and are responsive to IL-17 by polarizing towards a reactive phenotype and by producing several inflammatory cytokines and chemokines during EAE (67,(125)(126)(127). The impairment of IL-17-mediated signalling in astrocytes through the selective ablation of key signalling mediators was shown to ameliorate EAE by inhibiting the production of inflammatory chemokines, the infiltration of inflammatory cells (128,129) and the percentage of Th17 cells within CNS (130). For instance, in astrocytes, IL-17 induces the expression and production of IL-6 that, by acting in a positive feedback loop, may amplify Th17 cell differentiation (131)(132)(133). IL-17 also enhances the production of CCL20 in astrocytes (67,128), thus facilitating the recruitment of Th17 cells within CNS (90). Moreover, by up-regulating the expression of vascular adhesion molecule-1 (VCAM-1) on brain stem astrocytes, Th17-associated cytokines may further enhance the recruitment of both Th1 and Th17 cells within the CNS (134).
IL-17-mediated signalling in astrocytes also promotes the secretion of matrix metalloproteinases such as MMP-3 and MMP-9 that further compromise the integrity of the BBB favouring the recruitment of encephalitogenic T cells into the CNS (129,130).
Th17 cell-associated cytokines were also shown to affect the homeostatic functions of astrocytes. Kostic et al. reported that low doses of IL-17A impaired the ability of astrocytes to uptake glutamate from the extracellular space by reducing the expression of GLT-1 and GLAST transporters as well as of glutamine synthetase. In addition to reduce glutamate uptake, exposure of astrocytes to IL-17A also caused a Ca 2+ -dependent glutamate release, thus favouring excitotoxic damage ( Figure 1) (135).

Role of Th1-Like Th17 Cells on Astrocyte Functions in MS
Despite most of the studies carried out to investigate the crosstalk between inflammatory Th cells and astrocytes focused on Th1 and Th17 cells, the recent identification of highly pathogenic Th1-like Th17 cells in both EAE (49,136) and MS (136)(137)(138)(139)(140) suggests their contribution in promoting the morphological and functional changes occurring during astrogliosis. Th1-like Th17 produce TNF-a, GM-CSF, IL-17A, although at lower levels than Th17 cells, high levels of IFN-g, co-express CXCR3 and T-bet together with CCR6 and RORgt, and express IL-23R (122,141,142).
Most of the cytokines produced by Th1-like Th17 cells may exhibit synergistic detrimental effects on astrocytes. For instance, IFN-g and TNF-a cooperate with IL-17A by inducing the production of inflammatory chemokines in astrocytes and enhancing the recruitment of encephalitogenic T cells into the CNS (90,128,132,143). Moreover, IFN-g and IL-17A produced by Th1-like Th17 cells induce the up-regulation of intercellular adhesion molecule-1 (ICAM-1) and VCAM-1 in cortical astrocytes within CNS lesions during EAE (144). Finally, a detrimental contribution of Th1-like Th17 cells on astrocytes is strongly supported by the above cited effects of IFN-g in combination with GM-CSF. Indeed, these cytokines cooperate in impairing the production of neurotrophic factors as well as in promoting oxidative stress in astrocytes (90) and enhance astrocyte-dependent glutamate excitotoxicity induced by IL-17A ( Figure 1) (135). Nevertheless, further studies are required to elucidate this issue.

Crosstalk Between CD8 + T Cells and Astrocytes in MS
Although inflammatory Th cell subsets have long been regarded as the main effectors of MS pathogenesis, an important pathophysiological role of CD8 + T cells has also recently been recognized. Histopathological studies of immune cell infiltrates in post-mortem brain tissues from MS patients showed a prevalence of CD8 + T cells compared to CD4 + T cells (105,145). Moreover, sRNA-seq analyses revealed a prominent oligoclonal expansion of CD8 + T cells in the peripheral blood and CSF of MS patients (146,147).
Activated CD8 + T cells may contribute to BBB breakdown by inducing the activation of astrocytes and the downregulation of both occludin and claudin-5 in perforin-dependent and nonapoptotic manner (148). Once entered into the CNS, CD8 + T cells may be reactivated by MHC class I-expressing resident glial cells, including reactive astrocytes (145), thus contributing to tissue damage and neuroinflammation (149). In EAE, CD8 + T cells also produce IL-17 thus supporting Th17-mediated dysregulation of astrocyte functions (150). Astrocytes in turn may enhance the cytotoxic activity of CNS-infiltrating CD8 + T cells by producing IL-15 (151). Immunohistochemistry analysis of post-mortem brain tissues from MS patients revealed the expression of IL-15 in reactive GFAP + astrocytes located in both acute and subacute MS lesions as well as near blood vessels. The exposure of CD8 + T cells to astrocyte-derived IL-15 enhanced antigen-specific cytotoxicity as well as the expression of lytic enzymes (granzyme B and perforin) and natural killer group 2 member D (NKG2D) (151). NKG2D is a transmembrane receptor constitutively expressed on human CD8 + T cells that recognizes stress-induced ligands on target cells and enhances TCR-mediated cytotoxicity (152). Notably, ULBP4, a NKG2D ligand, was highly expressed on astrocytes in active and chronic active MS lesions from post-mortem brain tissues. Furthermore, the addition of soluble ULPB4 to CD8 + T cells from MS patients co-cultured with astrocytes enhanced the production of inflammatory cytokines and increased T cell motility, thus suggesting an important contribution of the crosstalk between CD8 + T cells and astrocytes in synergy with CD4 + Th cells to CNS damage in MS (153).

Impact of T Cell Targeting MS Therapeutic Drugs on Astrocytes
The crosstalk between inflammatory T cells and astrocytes in sustaining neuroinflammation and neurodegeneration in MS is supported by recent evidences that some of the diseasemodifying drugs target both inflammatory T cells (11) and astrocytes (154).
Glatiramer acetate (GA), an acetate salts composed of a mixture of four synthetic polypeptides (glutamate, lysine, alanine, and tyrosine), suppresses inflammatory Th1 cells by promoting their shift to an anti-inflammatory Th2 cell phenotype (155)(156)(157) and by increasing the frequency of suppressive regulatory T cells (Treg) (158). In EAE mice, GAspecific Th2 cells produced high levels of BDNF, IL-10 and TGFb following their adoptive transfer within CNS (159). Moreover, the production of immunosuppressive cytokines and neurotrophic factors by GA-specific T cells primed astrocytes to produce IL-10 and TGF-b (159), thus promoting their transition from a neurotoxic to a neuroprotective phenotype (160). More recent data from Eilam et al. also evidenced that GA treatment of EAE mice partially abrogated BBB disruption by increasing the expression of claudin-5 on astrocytes and by restoring their end-feet connections with the NVU (54).
Dimethyl fumarate (DMF) acts on T cells by reducing the total number of circulating T cells (161,162), in particular of IFN-g-and IL-17-producing Th cell subsets and CD8 + T cells (163,164), and by increasing the percentage of Th2 and Treg cells (165,166). DMF treatment of primary reactive astrocytes derived from both human and murine brains evidenced the ability of this drug to reduce the secretion of inflammatory cytokines and chemokines as well as to prevent the production of ROS (167) by promoting anti-oxidant gene expression (168). Similar effects were observed by treating human astrocytes with a novel fumarate, isosorbide-DMF (IDMF). Genome-wide expression analysis of human astrocytes treated with DMF and IDMF evidenced the ability of both compounds to downregulate the expression of several genes associated with neurotoxic reactive astrocytes, including MMP9, CCL2 and ICAM1 (169).
Fingolimod (FTY720) and siponimod (BAF312) are two antagonists of the sphingosine-1-phosphate receptor (S1PR) approved for RRMS and SPMS, respectively (11,170). Both drugs bind S1PR and induce its internalization, thus sequestering T cells in lymphoid organs and reducing circulating inflammatory CD4 + and CD8 + T cells (11,(170)(171)(172). In both human and murine astrocytes, fingolimod treatment was shown to impair the production of inflammatory and neurotoxic factors (173) and to promote the secretion of neurotrophic mediators (174). Moreover, Trkov Bobnar et al. showed that fingolimod reduced the expression of MHC-II on the surface of IFN-gtreated astrocytes, thus preventing their activity as APCs (175).
As fingolimod, siponimod was reported to impair inflammatory cytokine expression in human astrocytes and to restore astrocyte-endothelial cell connections by up-regulating the expression of claudin-5 and ZO-1 (176,177). Finally, in human astrocytes, both fingolimod and siponimod prevented glutamate neurotoxicity by restoring the expression of GLAST and GLT1 on astrocytes and glutamate uptake (177,178).
Laquinimod (LQ) is a quinoline-3-carboxamide derivate under clinical trial evaluation for the treatment of RRMS (179). In EAE, LQ treatment ameliorated disease progression by reducing the polarization and recruitment of Th17 cells as well as the production of inflammatory cytokines (180,181). Treatment of human astrocytes with LQ inhibited IL-1binduced downregulation of glutamate transporters GLAST and GLT1 and restored astrocyte glutamate uptake (178). Consistently, LQ treatment ameliorated EAE by suppressing, in astrocytes, the expression of inflammatory mediators such as IL-6 and ROS and by inducing a transcriptional program associated to homeostatic chemokines, neurotrophin, axonal guidance and transendothelial migration (182).

CONCLUSIONS
The contribution of Th1, Th17, Th1-like Th17 inflammatory cytokines in activating astrocytes to gain a neurotoxic phenotype is beginning to be studied in MS. However, astrocytes may also counteract inflammation by producing several factors, which reduce BBB breakdown, leucocyte infiltration and promote remyelination, axonal regeneration and neurogenesis (21). Therefore, therapeutic strategies aimed at counteracting the infiltration of inflammatory Th cell subsets into the CNS and at polarizing reactive astrocytes towards a neuroprotective phenotype may be beneficial to arrest disease progression as well as to stimulate repair processes. A deeper understanding of the cross-talk between T cells and astrocytes in MS will be seminal for the development of more efficient therapies damp ening infl ammato ry T ce lls a n d st i m u l at i n g neuroprotective astrocytes.

AUTHOR CONTRIBUTIONS
MK, CA, and VT wrote the original draft. MF contributed to reviewing and editing the manuscript. LT contributed to writing and editing the manuscript. All authors contributed to the article and approved the submitted version.


Toward Minimal Complexity Models of Membrane Reactors for Hydrogen Production

Membrane reactors are inherently two-dimensional systems that require complex models for an accurate description of the different transport phenomena involved. However, when their performance is limited by mass transport within the reactor rather than by the selective product permeation across the membrane, the 2D model may be significantly simplified. Here we extend results previously found for methane steam reforming membrane reactors to show that such simplified two-dimensional model admits either a straightforward analytical solution for the cross-section averaged concentration profile, or can be reduced to a 1D model with an enhanced Sherwood number, depending on the stoichiometry of the reaction considered. Interestingly, the stoichiometry does not affect the expression of the enhanced Sherwood number, indicating that a versatile tool has been developed for the determination of membrane reactor performance at an extremely low computational cost and good degree of accuracy.

Introduction
Membrane reactors represent an interesting solution for the decentralized production of high-purity hydrogen, as they allow the integrated production and separation of hydrogen in a single device. In addition, the removal of hydrogen as it is being produced allows to shift the equilibrium of the reaction toward the products, thereby increasing the overall conversion and allowing to carry out endothermic reactions at temperatures lower than those traditionally employed. This solution represents an advantage in terms of energy efficiency, but could also help avoid undesired reactions that take place at higher temperatures, such as coke formation in the presence of hydrocarbons. The use of these devices has been proposed and investigated for many applications, including the reforming of light hydrocarbons [1][2][3], the water-gas shift reaction [4,5], ammonia decomposition [6][7][8], and the dehydrogenation of alkanes [9][10][11]. The simplest reactor configuration envisaged is a tube-in-tube reactor, in which the reaction takes place in the annular volume filled with catalyst particles between the two concentric tubes. Heat may be provided through the outer wall of the tube. A hydrogen-permeable membrane is placed on the outer wall of the inner-most tube. Hydrogen is removed through the selective membrane and flows into the inner tube. A sweep gas is often employed in the permeate side to reduce the partial pressure of hydrogen and increase the driving force for permeation.
In the past years a significant evolution in modeling membrane reactors has been observed, from simple one-dimensional models [12,13] to more complex two-or threedimensional models [14,15]. While the former models are relatively simple to develop and can be solved at a low computational cost, they could be inaccurate even in predicting integral quantities [16,17]. In addition one-dimensional models seem to be conceptually inadequate for the description of membrane reactors, in which the transport of hydrogen takes place in the direction transversal to that of the main gas flow. Multi-dimensional models, on the other hand, allow to simultaneously account for several transport phenomena and could potentially be very accurate; however, the numerous parameters included in the more complex models are generally difficult to determine with accuracy and the effect of changes in the operating conditions or geometry are more difficult to grasp intuitively from a small set of results, because a single parameter could affect more than one phenomenon.
In this context, the development of one-dimensional models in which transversal concentration gradients are accounted for through a mass transfer coefficient is of interest, as it could combine the benefits of both one-and multi-dimensional models. The difficulty lies in the derivation of an expression for a mass transfer coefficient capable of accounting for the effects not only of convection and dispersion, but also of the reaction and of hydrogen permeation across the selective membrane. Previous experience on modelling membrane reactors for methane steam reforming had shown that, when the resistance to hydrogen permeation across the membrane is negligible, the behavior of the system may be described by a simplified 2D model [18,19]. Here we show that, for reactions characterized by simple stoichiometries, a straightforward analytical solution to the problem exists; whereas for more complex stoichiometries, it is necessary to introduce a mass transfer coefficient. Interestingly, it is found that the stoichiometry of the reaction considered does not affect the expression of the mass transfer coefficient. The present article is divided as follows. In Section 2 the problem is stated in greater detail, the modelling approaches are described in Section 3, and the results are presented in Section 4.

Statement of the Problem
Previous work on the description of membrane reactors for the production of hydrogen through the steam reforming of methane had highlighted that, depending on the operating conditions, the behavior of such systems could be limited by either radial mass transport within the packed bed or hydrogen permeation across the selective membrane. At a given inlet velocity, the transition between the two regimes was found to depend on the operating pressure. This is due to the fact that while the rate of the convective transport increases almost proportionally with pressure, the rate of permeation depends on the square root of pressure. In fact, hydrogen-selective membranes are generally Pd-based and the rate of permeation depends on the membrane permeability and the difference between the square roots of the hydrogen partial pressures in the retentate and permeate sides according to Sieverts' law [20] where J h is the molar hydrogen flux across the membrane, P m is the membrane permeability, p H 2 is the hydrogen partial pressure, and the superscripts r and p indicate the retentate and permeate sides, respectively. Under conditions in which the resistance offered by the membrane is negligible, the behavior of the reactor may be described through a simplified two-dimensional model. A more detailed explanation of these observations may be found in [19]. In [21], an expression for an enhanced Sherwood number for the evaluation of the hydrogen concentration profiles in steam reforming membrane reactors was developed, to further simplify the description of the problem to a one-dimensional model in which concentration gradients are accounted for through a mass transfer coefficient, or Sherwood number when the problem is analyzed in its dimensionless form. It is well known that the Sherwood number is a dimensionless group commonly employed to evaluate the mass transport coefficient, k y , between the bulk of a fluid, b, and a given surface, s and that it expresses the ratio between the rate of mass transport by convection and by diffusion (or dispersion). The Sherwood number is generally evaluated as a function of the Reynolds and Schmidt numbers; however, in reactive systems, the chemical reactions alter the local composition of the mixtures, thereby affecting the species concentration profile and hence the overall transport coefficient. In membrane reactors, there is the additional effect of the selective membrane. Consequently, correlations traditionally employed for the evaluation of the mass transfer coefficient lose significance. The aim of the present work has been to verify the possibility of extending the findings established in the conetext of steam reforming of hydrocarbons to different reactive systems, specifically to the dehydrogenation of propane. The range of operating conditions in which it is possible to describe the behavior of membrane reactors through this simplified approach with a good degree of accuracy was also investigated. In addition, the possibility of obtaining a simple analytical expression for the simplified 2D model was investigated for reactions characterized by a simple stoichiometry.
The problem was tackled through the following procedure 1. A simplified 2D model, derived in [19], was applied to the propane dehydrogenation reaction; 2.
an analytical solution to the average hydrogen concentration profile along the length of the reactor was obtained; 3.
an analytical expression for the enhanced Sherwood number was derived and employed in a 1D model of the membrane reactor; The model was developed for a tube-in-tube catalytic reactor configuration, in which the propane dehydrogenation reaction takes place Given its endothermicity, this reaction requires high temperatures and is prone to coke formation. To reduce both the energy cost and the risk of catalyst deactivation, it would be useful to lower the operating temperature; on the other hand, this would cause a reduction in the equilibrium conversion of propane. The problem could be solved by employing membrane reactors, in which hydrogen is selectively removed from the reacting volume as it is being produced, thereby shifting the equilibrium of the reaction.

Modelling
The 2D simplified model was developed under the following simplifying assumptions: where y i is the molar fraction of the i-th component; U is the axial velocity, which is constant following the assumption of constant density; D rr is the effective dispersion coefficient in the radial direction, considered to be the same for all components; ν i is the stoichiometric coefficient of the i-th component; r p is the volumetric rate of propane consumption; and the radial and axial coordinates are represented by r and z, respectively. The rate of propane dehydrogenation was expressed according to the correlation proposed by Sheintuch et al. [22] while neglecting the inhibiting effect on kinetics by propylene adsorption on the catalyst where k p is the kinetic constant, which, for a given catalyst, depends on temperature only; p C 3 H 8 is the partial pressure of propane, whereas the term (1 − η) accounts for the distance from chemical equilibrium conditions, with values ranging between 0 and 1 The boundary conditions are of fixed inlet composition, namely the equilibrium composition under the temperature and pressure conditions considered impermeability to the flux of all components on the outer wall impermeability of the inner wall to propane and propylene and hydrogen permeation according to Sieverts' law through the membrane placed on the outer wall of the inner tube In its dimensionless formulation, Equation (4) whereD rr is the dimensionless ratio between the effective dispersion coefficient in the radial direction and the molecular diffusion coefficient, D m , the Peclet number represents the ratio between the characteristic times of diffusion and convection, and the Damkholer number The dimensionless axial and radial coordinates were both defined on the basis of the characteristic length, l c , defined as the difference between the outer and inner reactor radii The boundary conditions remain virtually unchanged, with the exception of the one for hydrogen on the membrane wall, which becomes D rr Pe in which a dimensionless parameter representing the ratio between the characteristic times of convection and permeation, γ, is introduced having made the operating pressure dimensionless by defining it in terms of the atmospheric pressureP = P P atm (17) For thin annular channels it is possible to describe the problem in terms of Cartesian coordinates From here on, the dimensionless axial and transversal coordinates will be written as z and x, respectively, for easier readability.
As described in greater detail elsewhere [23] the problem described above may be simplified by considering an auxiliary variable, defined as a linear combination of the molar fractions of hydrogen and of the species from which it is produced, propane in the present case (19) for which the following balance equation holds and To be solved with the boundary conditions The latter boundary condition is a consequence of having considered negligible resistance to the transport of hydrogen across the membrane, from which one can set a y H 2 = 0 on the membrane wall, and local chemical equilibrium, a condition that can be met only if the partial pressures of hydrogen and propane both go to zero when approaching the membrane wall. The above homogeneous problem admits the following analytical solution where λ l = π 2 (2l + 1). Keeping in mind that, given the impermeability of the membrane to all components other than hydrogen, the Sherwood number, defined in Equation (2), is given by where and where y H 2 (z) is the average molar fraction of hydrogen and may be determined by solving the following set of equations Given the simple stoichiometry of the reaction, the above set of equations admits the analytical solution Having obtained the above solution, it is interesting to determine how such an average concentration profile would differ from the one obtained with a 1D model with an enhanced Sherwood number and to compare the expression of Sh(z) obtained from the propane dehydrogenation reaction with the one previously obtained for methane steam reforming. Figure 1a,b show the Sherwood number along the length of the reactor, evaluated according to Equation (28) at atmospheric pressure, temperatures between 400 and 700 • C and at Pe values of 10 and 100, respectively. With regards to the results shown in Figure 1b, it is interesting to note that at 400 • C, the Sherwood number remains almost constant along the entire reactor length. This is mainly attributable to the fact that at low temperatures, the equilibrium conversion of propane is lower than 10%, meaning that the hydrogen concentration is low and the flux permeating across the membrane is not sufficiently high to significantly increase the degree of propane conversion. The results show that, as in the case of the methane steam reforming reaction, the variation of the Sherwood number along the reactor axis may be described through an expression of the kind Sh(z) = Sh 0 z n

Results
at high values of the Peclet number, whereas the trend for Sh changes along the length of the reactor when the Peclet number is low. The different behavior observed depending on the value of Pe was expected because, when all other conditions are kept constant, increasing the value of Pe corresponds to a decrease in the dispersion coefficient; consequently, the performance of the reactor is increasingly limited by mass transport within the packed bed, i.e., the main assumption under which the simplified 2D model was developed. We now move on to consider the same approximated expression for Sh(z) as the one employed for the methane steam reforming reaction, namely [21] with and z * 1, and determine the average hydrogen concentration profiles by solving the following problem Figure 2 shows a comparison between the values of the Sherwood number along the length of the reactor, evaluated from the simplified 2D model and from Equation (33). The agreement is very good, particularly if taking into account that the approximate expression was simply predicted from results previously obtained for the methane steam reforming reaction.   It is clear that the agreement between the trends predicted with the two models is excellent, even though the expression for the Sherwood number was not developed specifically for this reactive system. The results also show that there is an error in the evaluation of the hydrogen molar fraction close to the reactor inlet, which causes the discrepancy between the two sets of results. On the other hand, the hydrogen recovery, defined as the ratio between the flowrate of hydrogen permeated across the membrane and the inlet flow rate of hydrogen, evaluated from the two models coincides, as shown from the results reported in Figure 4. This indicates that the proposed solution can be successfully employed for the evaluation of integral quantities at a very low computational cost.

Conclusions
A simplified 2D model, initially developed to describe the behavior of a membrane reactor for the steam reforming of methane was applied to the propane dehydrogenation reaction in the same type of device. It was shown that, due to the simple stoichiometry of the reaction considered, it is possible to obtain a straightforward analytical expression for the average hydrogen concentration profile along the length of the reactor. Although, in comparison to two-dimensional models, this solution does not allow to have detailed information on the transversal concentration profiles formed within the reactor, significant information is obtained at almost no computational cost. In addition, an attempt was made to describe the performance of the system through a 1D model making use of the same expression for an enhanced Sherwood number previously obtained for the methane steam reforming reactor, with the aim of validating the previously proposed approach. An excellent agreement was obtained between the trends of the average hydrogen concentration profiles along the reactor under conditions in which the behavior of device is limited by mass transport across the reactor bed. The results show that it is possible to evaluate integral quantities with a good degree of accuracy at very low computational cost. The possibility of employing the same expression for Sh(z) regardless of the reactive system indicates that the tool developed is extremely versatile.