Influence of the irradiated pulmonary microenvironment on macrophage and T cell dynamics.

Abstract

Background

The lung is sensitive to radiation, increasing normal tissue toxicity risks following radiation therapy. Adverse outcomes include pneumonitis and pulmonary fibrosis, which result from dysregulated intercellular communication within the pulmonary microenvironment. Although macrophages are implicated in these pathogenic outcomes, the impact of their microenvironment is not well understood.

Materials and methods

C57BL/6J mice received 6Gyx5 irradiation to the right lung. Macrophage and T cell dynamics were investigated in ipsilateral right lungs, contralateral left lungs and non-irradiated control lungs 4-26wk post exposure. Lungs were evaluated by flow cytometry, histology and proteomics.

Results

Following uni-lung irradiation, focal regions of macrophage accumulation were noted in both lungs by 8wk, however by 26wk fibrotic lesions were observed only in ipsilateral lungs. Infiltrating and alveolar macrophages populations expanded in both lungs, however transitional CD11b+ alveolar macrophages persisted only in ipsilateral lungs and expressed lower CD206. Concurrently, arginase-1+ macrophages accumulated in ipsilateral but not contralateral lungs at 8 and 26wk post exposure, while CD206+ macrophages were absent from these accumulations. While radiation expanded CD8+ T cells in both lungs, T regulatory cells only increased in ipsilateral lungs. Unbiased proteomics analysis of immune cells revealed a substantial number of differentially expressed proteins in ipsilateral lungs when compared to contralateral lungs and both differed from non-irradiated controls.

Conclusions

Pulmonary macrophage and T cell dynamics are impacted by the microenvironmental conditions that develop following radiation exposure, both locally and systemically. While macrophages and T cells infiltrate and expand in both lungs, they diverge phenotypically depending on their environment.

Introduction

The lung is sensitive to radiation injury limiting the dose that can be safely delivered for oncologic radiotherapy[1–3]. The pathology of radiation induced lung injury (RILI) is multifaceted, involving immediate direct injury to multiple lung-resident cell types along with induction of a progressive long-lasting inflammatory response[4]. This failure to resolve inflammation and return to normal tissue quiescence leads to long-term pulmonary dysfunction. Acute injury responses to radiation occur days to several weeks post exposure, while chronic inflammation, described clinically as acute pneumonitis, and late fibrotic remodeling, known as radiation induced pulmonary fibrosis (RIPF) develop months to years after radiotherapy[5, 6].

After radiotherapy, a perpetual innate immune response leads to chronic proinflammatory and profibrotic microenvironments involving cytokine signaling, macrophage recruitment, and disrupted communication between resident cell types that make up the lung niche[7–11]. A key component, alveolar macrophages adapt their function according to signals encountered in their microenvironment[12] to maintain homeostasis and orchestrate inflammatory responses, including injury resolution, tissue repair, and antigen presentation to T cells[13–16]. Macrophages that accumulate in the lung following radiation exposure are implicated in the development of chronic inflammation and RIPF[6, 17–19]. This is evidenced by our prior work and others, which finds that restricting the infiltration of monocyte-derived macrophages into the lung limits collagen accumulation and fibrotic remodeling, attenuating RIPF development[20–24]. Since these recruited macrophage populations have not been directly exposed to radiation, their pro-fibrotic programming is likely attributable to signals originating from the irradiated lung microenvironment[25]. While it is well known that macrophages contribute to the establishment of fibrotic disease, the contribution of T cells to this process is less well understood[26–28]. Although the T regulatory cell marker Foxp3 is associated with idiopathic pulmonary fibrosis[29], it appears that Foxp3+ cells differently affect this process at different phases of disease progression[30].

Immune cells are recruited to sites both inside and outside the field of irradiation where their functions are influenced by their microenvironment. Activation in a manner that is immunostimulatory is beneficial to promote tumor eradication. Indeed, combined use of immune checkpoint inhibitors along with radiotherapy has proven very effective in the treatment of NSCLC[31]. However these immune effects can also augment the direct cytotoxic effects of radiation therapy[32]. Moreover, cells directly exposed to radiation release danger signals to non-exposed cells outside the field, leading to abscopal effects distant to the site of irradiation[33]. While these out of field immune-mediated events can have therapeutic benefit in terms of tumor cytotoxicity, they can also result in adverse events and normal tissue toxicities[33–35]. We hypothesized that alteration of the pulmonary microenvironment by radiation exposure will affect both the presence and phenotype of the immune cell subsets within it. To develop a better understanding of the mechanisms responsible for immune-related toxicities we used a novel uni-lung irradiation murine model which enabled us to elucidate the influence of the pulmonary microenvironment in immune cell dynamics after irradiation.

Materials and Methods

Animals

C57BL/6J mice (female and male, 6-8 wks of age) obtained from Jackson Laboratory (Bar Harbor, ME) were housed five per cage in microisolator units under pathogen-free conditions and fed standard laboratory diet and water ad libitum. Mice were acclimated for one week prior to experimentation. The University Committee on Animal Resources approved all animal protocols.

Uni-lung Irradiation

Mice were irradiated using CT-guidance with a SARRP X-irradiator (Small Animal Radiation Research Platform; XStrahl Inc., Suwanee, GA; dose rate of 3.1Gy/min). An opposing 10mm x 10mm collimation two-beam set-up was positioned in a sagittal orientation to ensure full coverage of the right lung while avoiding off target tissue exposure, such as the heart and thymus (Figure 1A). A fractionated dose of 6Gyx5 was used. Control animals were sham-irradiated with identical handling. Dose Volume Histograms (DVHs) were used to confirm no dose was delivered to off target tissue (Figure 1B).

Figure 1.

SARRP image guiding and focal x-ray irradiation of right lung. (A) CT image for beam set up and subject illustrating placement of irradiated field. (B) Dose Volume Histogram shows right lung received 100% of the dose. Off target left lung thymus and heart show no dose. (C) Scheme illustrating the 3 conditions under comparison. Targeted (ipsilateral) right lungs are designated as RT – ipsi. Off-target (contralateral) left lungs are designated as RT – contra. Control left lungs from mice not receiving radiation are designated as None. Subject’s right and left are depicted from observational point of view.

Sample Collection, Magnetic Cell Sorting and Flow Cytometry

Ipsilateral, contralateral and no-irradiation control lungs (Figure 1C) were collected from animals at 8, 16 and 26 wk following irradiation. At the 8 wk time point CD45+ myeloid cell enriched lung digests were generated as previously described[21]. Aliquots of 1×10⁶ cells were transferred to staining buffer (PBS plus 10% FBS; BD Pharmingen, San Jose, CA) for flow cytometric analysis or to PBS for proteomics analysis. Surface staining for flow cytometry was performed as previously described[21] using antibodies described in supplemental methods. Data were analyzed using FCS Express software (De Novo Software, Pasadena, CA).

Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomics.

CD45+ cells were washed with inhibitor cocktails (Sigma, P 5726 and P 0044, per manufacturer instruction) and were extracted from pellets using the MPLEx method[36] as previously described for pulmonary samples[37]. Protein extractions were performed as previously described and LC-MS/MS runs were performed on a Q-Exactive Plus mass spectrometer[38]. MaxQuant Label free quantification was used and data processing and statistics were performed using the homemade package RomicsProcessor v1.0.0[39] (https://doi.org/10.5281/zenodo.3956544 ). Data analysis code was deposited on Github and raw LC-MS/MS data is publicly available on MassIVE FTP at the following address https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=f2900399a89e4ffda7f0e1eda3bbf84d. See supplemental methods for a complete description.

Histology and Immunohistochemistry

Lungs, collected at 8 and 26 wk following irradiation, were inflation-fixed and paraffin embedded. Lung sections (6 μm) were stained with Hematoxylin and Eosin or Gomori’s trichrome (Leica Biosystems Richmond, Inc., Richmond, IL) and immunohistochemical analysis performed as previously described[40]. Antibody binding was performed with Mannose Receptor (CD206) (1:2500, Abcam, Cambridge, UK), Arginase-1 (1:300, Proteintech Group, Chicago, IL), monocyte chemotactic protein-1 (MCP-1; CCL2) (1:1000, Abcam), or CD3 (1:100, Abcam) and visualized by horseradish peroxidase/diaminobenzidine detection. Images were acquired on an Olympus BX51 microscope (Olympus America, Center Valley, PA).

Statistical Analysis and Pathway Enrichment

Data, expressed as mean ± SEM, were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. Differences were considered significant at P < 0.05. Analysis was performed using GraphPad Prism 9 (GraphPad Software Inc, La Jolla, CA). Differentially expressed protein lists were then analyzed using the ToppFun webapp (https://toppgene.cchmc.org/enrichment.jsp) and select GO: Biological Process terms were reported.

Results

A uni-lung treatment methodology was used to determine how macrophages were recruited to the ipsilateral and contralateral lungs after targeted pulmonary irradiation, and how radiation injury changes the pulmonary microenvironment, both locally and systemically (Figure 1). Timepoints examined capture early, acute and late radiation responses. Alveolar and infiltrating macrophage subsets were identified (Figure 2A; description of gating scheme provided in supplemental text). Both subsets increased in both ipsilateral and contralateral lungs by 8wk post exposure, with increased alveolar macrophages persisting to the 16wk timepoint (Figure 2B, C). Additionally, the percentage of infiltrating macrophages in the contralateral lung was significantly increased relative to the ipsilateral lung at this timepoint. This demonstrates macrophage trafficking into the lungs was irrespective of direct radiation exposure. Newly-differentiated mature alveolar macrophages that derive from infiltrating macrophages are functionally active and maintain expression of CD11b[41]. These CD11b+ alveolar macrophages were identified and were significantly increased in ipsilateral but not contralateral lungs until 26wks, when increases were observed in both lungs of irradiated mice (Figure 2D). Radiation also impacted macrophage phenotype in both lungs of irradiated mice. Expression of the pro-inflammatory marker Ly6C significantly increased in infiltrating macrophages in both lungs at 4wk and 16wk post exposure (Figure 2E) suggesting a response that was independent of the local microenvironment. In contrast, Ly6C was nearly absent in the resident alveolar macrophage population (less than 2% positive), but was increased by radiation exposure in CD11b+ alveolar macrophages in contralateral lungs through 8wk (Supplementary Figure 1). Macrophage mannose receptor (CD206) is indicative of pro-resolution and wound healing phenotypes and is expressed on lung macrophages but not monocytes[42]. The vast majority of alveolar macrophages were CD206+, irrespective of radiation exposure or timepoint (Supplementary Figure 1), but infiltrating macrophages from both lungs increased this marker at 8wk, and this was maintained in ipsilateral lungs at 16wk (Figure 2F). In CD11b+ alveolar macrophages CD206 expression differed between ipsilateral and contralateral lungs. It increased in contralateral lungs at 4wk and 8wk but decreased in ipsilateral lungs at 16wk and 26wk (Figure 2G).

Figure 2.

Macrophage dynamics following un-lung irradiation. Macrophage subpopulations and phenotype was analyzed from lung digests collected from female C57Bl/6J mice exposed to either 0 or 6Gyx5 right lung irradiation. (A) Gating scheme. (B) Percentage of infiltrating macrophages (Inf.; CD45+ autofluorescent−F4/80+ Ly6G−CD11b+ CD11c−). (C) Percentage of alveolar macrophages (AMs; CD45+ autofluorescent+ F4/80+ Ly6G−CD11b+/− CD11c+). (D) Percentage of AMs that are CD11b+. (E) Percentage of Inf. that are Ly6C high. (F) Percentage of Inf. that are CD206+. (G) Percentage of CD11b+ AMs that are CD206+. Each bar represents the mean + SEM (n=3-4 mice/treatment group/time point). *Significantly different comparisons (P < 0.05) identified by brackets.

RILI develops in a heterogenous fashion, forming discreet lesions consisting of accumulated macrophages and other inflammatory cells and, at sufficient radiation doses, fibrotic remodeling. To differentiate macrophages responses occurring within these pathologic regions from those in surrounding tissue, a separate cohort of animals designated for histological evaluation received identical radiation exposures alongside those selected for flow cytometry analysis. Accumulations of macrophages were detected in both ipsilateral and contralateral lungs at 8wk, a finding not present in unexposed control lungs (Supplemental Figure 2). RILI was observed in ipsilateral lungs by 26wk, where lymphocytes and enlarged macrophages accumulated in collagen dense lesions located on the periphery (Supplemental Figures 2,3). To extend the flow cytometry findings, macrophage expression of CD206 and the pro-fibrotic alternative activation marker arginase-1 were evaluated. At 8wks macrophages in control lungs stained positively for CD206 but did not express arginase-1 (Figure 3). Accumulated macrophages in both ipsilateral and contralateral lungs of irradiated subjects were arginase-1+, while CD206+ macrophages were absent from ipsilateral but not contralateral lungs. By 26wk, RILI lesions in the ipsilateral lung had a subset of large arginase-1+ macrophages that lacked CD206 expression (Figure 4). In contrast, arginase-1+ cells were absent outside areas of RILI in ipsilateral lungs and in contralateral and control lungs, while CD206+ macrophages were present. We next investigated the presence and location of cells expressing CCL2/MCP-1, a chemokine that regulates infiltration of inflammatory monocytes and macrophages. At 8wks, some MCP-1+ lymphocytes, but not macrophages, were present within inflammatory cell accumulations in ipsilateral and contralateral lungs, while non-irradiated control lungs showed no staining (Figure 3). At 26wk, a subset of cells within areas of RILI in the ipsilateral lung were MCP-1+ (Figure 4). This was also observed in surrounding tissue and the contralateral lung. Control lungs showed weak staining in some but not all macrophages.

Figure 3.

Uni-lung irradiation affects macrophage phenotype in both lungs. Macrophage phenotype was analyzed within lungs collected from female C57BL/6J mice exposed to either 0 or 6Gyx5 right lung irradiation. Lung sections were prepared at 8 wk following exposure. Immunohistochemistry was performed using antibodies to Arg-1, CD206 and MCP-1 and visualized using DAB. Original magnification, 400x. Representative images are shown from n=3–4 mice/treatment group/time point.

Figure 4.

Uni-lung irradiation affects macrophage phenotype in both lungs. Macrophage phenotype was analyzed within lungs collected from female C57BL/6J mice exposed to either 0 or 6Gyx5 right lung irradiation. Lung sections were prepared at 26 wk following exposure. Immunohistochemistry was performed using antibodies to Arg-1, CD206 and MCP-1 and visualized using DAB. Original magnification, 400x. Representative images are shown from n=3–4 mice/treatment group/time point.

In order to identify how focal irradiation affects immune cells in ipsilateral and contralateral lungs relative to non-irradiated lungs, at 8wks we performed unbiased proteomics analysis on isolated CD45+ cells, using aliquots of the same digests analyzed by flow cytometry. The numbers of significantly differentially expressed proteins between each lung condition were identified using three paired t-test analyses, each comparing results from two experimental conditions (Figure 5A). There were 87 proteins differentially expressed between the ipsilateral and contralateral immune cells, 93 differentially expressed proteins between the ipsilateral and non-irradiated lung immune cells, and 93 differentially expressed proteins between the contralateral and non-irradiated lung immune cells. We next compared the protein lists from each individual t-test against each other. Of these proteins, 12 proteins were common between the ipsilateral versus contralateral & ipsilateral versus non-irradiated comparisons. We determined that 18 proteins were shared in ipsilateral versus non-irradiated & non-irradiated versus contralateral. Additionally, there were 13 proteins differentially expressed in the ipsilateral versus contralateral & non-irradiated versus contralateral groups. Heatmaps of these proteins were generated (Figure 5B, selected proteins of interest are listed in brackets to the left of each heatmap) and pathway analysis performed (Figure 5C, selected pathways of interest listed). A list of overrepresented proteins found in each selected pathway is displayed in Supplemental Table 1. Supplemental Figure 4 contains plots for selected proteins depicting the normalized intensity in each of the experimental conditions. As expected, many inflammatory proteins were found at higher levels in immune cells isolated from irradiated lungs as compared to immune cells isolated from either non-irradiated or contralateral lungs. Conversely, many proteins from immune cells were detected at lower levels in contralateral lungs versus non-irradiated lungs. The three experimental groups were also compared by ANOVA which identified similar Gene Ontology pathways as from the t-test analyses (Supplemental Figure 5). Pathway analysis then provided additional insight. Common to all comparisons, pathway analysis returned alterations in viral processes, while pathways related to T cell signaling, leukocyte activation and macrophage regulation were detected individually in the 3 comparisons.

Figure 5.

Uni-lung irradiation affects the proteasome in both lungs. Proteomics analysis was performed on lung digests collected from female C57BL/6J mice exposed to either 0 or 6Gyx5 right lung irradiation. Lung digests were analyzed at 8 wk following exposure. (A) The number of significantly differentially expressed proteins for comparisons between each of the 3 experimental conditions was determined. RT-ipsi vs none is depicted in peach, RT-ipsi vs RT-contra is depicted in green, and RT-contra vs none is depicted in blue. (B) Heat maps (with select proteins of interest listed) depict significantly differentially expressed proteins for each comparison. (C) Pathway analysis (with select pathways of interest listed) was performed for each comparison between the 3 experimental conditions. n=3–4 mice/treatment group. Comparisons where P < 0.05 were considered to be significant.

Since pathway analysis identified radiation-induced alterations in processes related to macrophage and T cell interaction, lung T cell populations were assessed by flow cytometry in the same lung digests used for macrophage analysis (Figure 6). Radiation did not significantly alter the total percentage of T cells overall (identified as CD3+) except for a transient increase in ipsilateral lungs at 8wks (Figure 6A). However, within all CD3+ T cells, CD8+ T cells significantly increased in ipsilateral lungs at 8wk and 16wk post exposure and in contralateral lungs at all time points (Figure 6B). CD4+ T cells were increased in both lungs of irradiated mice at 8wks, before returning to control values by 16wk and were significantly decreased in contralateral lungs by 26wk (Figure 6C). Radiation increased the percentage of Tregs in ipsilateral lungs at all time points, and in contralateral lungs at 8wk post exposure (Figure 6D). Histological evaluation of T cell accumulation within the lung revealed CD3+ cells within inflammatory cell accumulations in ipsilateral lungs by 8wk post exposure, while in contralateral and non-irradiated control lungs sparse staining was observed (Figure 6E). At 26wk, a subset of CD3+ cells were present in lymphocytic accumulations within areas of RILI in ipsilateral lungs, but not contralateral or control lungs.

Figure 6.

Uni-lung irradiation affects T cell dynamics in both lungs. T subsets were identified within lungs collected from female C57BL/6J mice exposed to either 0 or 6Gyx5 right lung irradiation. Lung digests were collected at 8, 16 and 26 wk following exposure. (A) Following doublet and dead cell exclusion, T cells were identified as CD45+ CD3+ autofluorescent− F4/80− Ly6G− (B) Within CD3+ T cells, CD8+ cells and (C) CD4+ cells were identified. D Within the CD4+ population T regulatory cells (T reg) were identified as Foxp3+ CD127−. Each subset is represented as the percentage of CD45+ cells per whole lung. Each bar represents the mean + SEM (n=3-4 mice/treatment group/time point). *Significantly different comparisons (P < 0.05) identified by brackets.

Discussion

Using a novel uni-lung irradiation approach, - we show that pulmonary macrophage dynamics are impacted by microenvironmental conditions that develop in the lungs following radiation exposure. This in turn influences their differentiation, activation state and function. Our findings demonstrate that while macrophages infiltrate and expand in both lungs, they diverge phenotypically depending on their irradiated environment, such that differences exist between the ipsilateral and contralateral lungs, which also both differ from naive non-treated control lungs. Resident alveolar macrophages and infiltrating inflammatory monocyte-derived macrophages have distinct ontology[43] and can be differentiated respectively as CD11c^highCD11b^low and CD11b^highCD11c^low[41]. As the infiltrating population enters the lung, signals from the pulmonary microenvironment induce differentiation, to a phenotype similar to resident macrophages, including upregulation of CD11c[20]. Duan et al. and others have identified that an intermediate population of CD11b+ alveolar macrophages appear in the lung under inflammatory conditions[41]. These cells express markers characteristic of resident alveolar macrophages (autofluorescent+/CD11c+), but originate from the bone marrow, demonstrating that this subset has infiltrated into the lung and is transitioning into a more resident-like, differentiated phenotype[41, 44]. Our findings also identify this CD11b+ alveolar macrophage population, and reveal that it is persistently expanded primarily in the directly exposed ipsilateral lung. This could reflect that radiation-altered microenvironmental factors disrupt macrophage differentiation causing this transitory subset to linger in the directly exposed lung. The possibility that this observation could merely be the result of greater cell influx is not supported by our findings since the infiltrating population did not predominate in the ipsilateral lung. Additionally, these cells are not directly affected by radiation exposure, since CD11b+ identifies this subset as originating from outside the lung. Interestingly, expanded populations of both infiltrating macrophages and CD11b+ alveolar macrophages in the contralateral lung by 26wk raise the possibility that systemic effects result from the chronic pathology occurring at this time. Histological evaluation confirmed flow cytometry findings and provided additional locational insight, showing macrophage accumulation into both lungs of irradiated subjects at 8wk and correlated the development of RILI in ipsilateral but not contralateral lungs to the preferential expansion of CD11b+ alveolar macrophages in the ipsilateral lung. This is further supported by prior work by our group and others which finds that restricting the infiltration of monocyte-derived macrophages into the lung limits collagen accumulation and fibrotic remodeling[20, 21, 45].

Although it is known that radiation injury results in the initial accumulation of pro-inflammatory M1 macrophages followed by pro-resolution M2 macrophages at later stages of the progression[46, 47], how the microenvironmental conditions impact macrophage phenotype has not been fully explored. The impact of the microenvironmental conditions is now defined in this study using our uni-lung model. CD206 is expressed by alveolar macrophages during quiescence and during inflammation resolution and return to homeostasis[48], while arginase-1 has been linked to pro-fibrotic programming[49]. Increased CD206 expression was transient and occurred in the less differentiated infiltrating population and in CD11b+ alveolar macrophages residing in contralateral lungs, possibly reflecting more productive phenotypic changes occur in this microenvironment. However, in the directly irradiated ipsilateral lung at late timepoints corresponding to the fibrotic phase, decreased CD206 expression within CD11b+ alveolar macrophages was accompanied by absence of macrophages staining for this marker in pathologic areas of RILI and the appearance of enlarged arginase-1+ macrophages. Coupled with the fact that RIPF is restricted to the field of irradiation, this further implicates these cells as being aberrantly activated and likely contributors to pro-fibrotic processes. Others have reported high arginase-1 expression in CD11b+ alveolar macrophages that were isolated following inflammatory conditions, including during chronic inflammation and fibrotic remodeling[44]. Notably this isolated population also expressed high levels of pro-inflammatory iNOS and pro-fibrotic IL-10. By contrast, radiation-induced increases in the pro-inflammatory markers Ly6C and MCP-1 were not restricted to directly irradiated lungs, suggesting that classically pro-inflammatory macrophage phenotypes may be less dependent on local microenvironmental signals, and contribute to macrophage pools within both irradiated and contralateral lungs.

T cells also influence the development of RIPF by contributing to inflammation and repair responses[50]. In the ipsilateral lung, T cells present in areas of macrophages accumulation where intercellular communication can occur coupled with expanded CD4+ T cells and Treg populations points to the influence of additional radiation-induced effects on microenvironmental factors. This could be either by directly affecting T cells or by influencing the behavior of the alveolar macrophages they interact with. For example, macrophages can promote the generation of Tregs through expression of TGF-beta, a cytokine involved in pro-fibrotic signaling[15]. Furthermore, the expansion of CD8+ T cells in both lungs of exposed mice and the persistence of this population in the contralateral lung to 26wk demonstrates lasting systemic effects on this population. This could have negative outcomes on regulation of immunity and control of future insults to this lung.

Unbiased proteomics analysis of differentially expressed proteins by lung immune cells also showed both local and systemic responses to radiation and revealed that pathways related to immune function were impacted. Not surprisingly, many proteins were detected at higher levels in immune cells isolated from the ipsilateral lung. Of interest, we found that CD169 and legumain, both primarily expressed by macrophages and more specifically upregulated in M2 macrophage subsets in the lung during inflammatory and fibrotic conditions[51, 52], were increased in irradiated ipsilateral lungs when compared to either contralateral or control lungs. This is in line with our findings of an accumulation of alternatively activated arginase-1 positive macrophages specific to the ipsilateral lung, as well as the greater expansion of CD206+ infiltrating macrophages in these lungs at the 8 wk time point by flow, which are the same samples that were analyzed for proteomics. Furthermore, macrophage overexpression of CD169 has been shown to promote the accumulation of T regulatory cells[53], a response we also observed in the ipsilateral lungs at the 8 wk time point. Other proteins upregulated when comparing ipsilateral versus no-irradiation immune cells, include the High affinity immunoglobulin epsilon receptor subunit gamma, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, H-2 class I histocompatibility antigen D-B alpha chain, Cathepsin Z, and Heme oxygenase 2. Downregulated proteins include Catalase, CD166, and Catenin beta-1. Pathway analysis revealed that proteins belonged to pathways involved in metabolism (fatty acids, reactive oxygen species, and generation of energy), apoptosis, leukocyte activation/proliferation, and protein localization to the endoplasmic reticulum. In comparing immune cells from ipsilateral versus contralateral lungs, proteins that were expressed at higher levels include Legumain, Toll-interacting protein, H-2 class II histocompatibility antigen A beta chain, H-2 class I histocompatibility antigen D-B alpha chain, and Rho-related GTP-binding protein RhoG. The increased expression of these proteins suggests the continual promotion of a pro-inflammatory microenvironment in the targeted lung. Proteins belonged to pathways including TLR and Fc signaling, histone ubiquitination, viral processing, and macrophage function. Comparing contralateral and no-irradiation lungs, pathways revealed included antigen presentation, cell cycle, pro-inflammatory/antigen receptor signaling, protein trafficking, and proteosome-mediated protein degradation. Although some proteins such as Beta-2 Microglobulin, Ubiquitin-conjugating enzyme E2 N, and Phosphatidylinositol 4-kinase alpha were found at higher levels in immune cells from contralateral lungs vs no-irradiation lungs, the vast majority of proteins were at lower levels in contralateral lungs, which included proinflammatory and cytotoxic proteins (e.g., Ceruloplasmin, Calumenin and Complement 3), proteins involved in antigen presentation (e.g., 26S proteasome regulatory subunit 7, 26S proteasome regulatory subunit 8, 26S proteasome non-ATPase regulatory subunit 2, and 26S proteasome non-ATPase regulatory subunit 6). These systemic effects in contralateral lungs would be predicted to impair mounting an effective adaptive immune response. This could have serious implications for patients who receive radiation therapy, as these changes lead to the prediction that their ability to mount effective immune responses against pathogens would be impacted. This is evidenced by reports of increased susceptibility to infection, including to SARS-CoV-2, in patients that have undergone chemotherapy and radiotherapy[54, 55]. Supplemental Table I highlights additional specific proteins that were differentially expressed in the ANOVA analysis.

An additional benefit of this model is the specificity of radiation exposure to a contained field. Due to the exclusion of heart and thymus in the field of exposure, this study provides further evidence that these observations were not due to the exposure of non-pulmonary tissue, as could be the case in whole thorax exposure models[56]. Although our findings are focused on macrophage dynamics, it is likely that many lasting perturbations will impair lung homeostasis. For example, persisting radiation-induced vascular dysfunction would affect leukocyte trafficking and therefore alter the pool of macrophages and other immune cells present in the lung[57]. Another important aspect that is not explored in this study is the effect of mouse strain on sensitivity to developing late effects[58–60] as well as differences in macrophage dynamics, since their activation state and phenotype in response to radiation exposure are known to vary between pneumonitis vs fibrosis susceptible strains[11, 17, 61].

In patients receiving radiation therapy to the chest area, exposure of normal tissue within the lung is sometimes unavoidable, causing lasting impacts on immune function, both locally and systemically, potentially altering immune responses to subsequent exposures. This has implications for patients receiving a subsequent course of immunotherapy, as alterations that would impair the control of inflammation may negatively impact responsiveness to treatment[62, 63]. For example, an enhanced immunosuppressive environment may increase the likelihood of metastasis. Alternatively, increased CD8+ T cells may enhance tumor control, but increased arginase-1 may help cancer cells evade immunity[64]. Additional experiments are needed to fully assess the impact of a tumor on lung microenvironmental factors. Our data showing that 8 weeks post-irradiation, immune cells in the contralateral lung express lower levels of proteins involved with antigen presentation suggest the contralateral lung may exhibit a degree of immunosuppression. Conversely, immune cells from ipsilateral lungs express more proteins associated with activation which is consistent with increased inflammation. Future studies will explore beneficial immunotherapy strategies with the goal of identifying novel methods to target macrophage responses in favor of productive resolution of inflammation, thus preventing inappropriate inflammation and fibroblast activation.

Highlights.

• Alveolar macrophages are a key component of the lung microenvironment and alter their functionality according to signals they encounter.

• Macrophages accumulate in the lung following exposure to ionizing radiation and are implicated in chronic inflammation and pathogenesis of radiation induced pulmonary fibrosis

• A novel uni-lung irradiation approach allows for investigation of the role the pulmonary microenvironment plays in macrophage and T cell dynamics in murine models.

• Findings from this study contribute to a better understanding of the mechanisms responsible for immune-regulated radiation toxicities to the lung, both locally and systemically.