Toxics, Vol. 10, Pages 797: T Cells Contribute to Pathological Responses in the Non-Targeted Rat Heart following Irradiation of the Kidneys

1. IntroductionHeart disease is a significant detrimental event caused by radiation therapy for some cancers [1]. The pathogenesis of radiogenic heart disease is not well defined since the heart is not always directly in the irradiated field. Identifying the origins of radiogenic heart disease will enable better therapies to be developed. Irradiation of the organs below, but not above, the diaphragm with 10 Gy of X-rays in wild-type WAG/RijCmcr (WAG) rats causes fibrotic heart disease that is quantitatively and qualitatively similar to that seen following total body irradiation [2]. These findings suggest local radiation can exert a systemic response to cause non-targeted, i.e., abscopal effects. Non-targeted effects are effects manifest in non-irradiated cells that received signals communicated from an irradiated cell [3].Targeted irradiation of the kidneys with 10 Gy in wild-type WAG rats causes mature T cells, macrophages and natural killer cells to infiltrate the cortex and medulla 120 days after exposure [4], though the response of the immune system at an earlier time point remained undefined. Both human [5,6] and rat [4] kidneys contain myeloid and lymphoid cells that may respond to local damage from radiation and thereby determine whether these insults are resolved or progress and reach beyond the irradiated organ. To examine the role of T cells in the pathogenesis of radiation nephropathy, we initially chose a genetic model of immune cell deficiency that lacks functional T cells using the Dahl S rat [7]. In a local kidney irradiation model, we have shown significantly less renal radiation injury of genetically T cell knock down Dahl S rats [4]. This supports a role for the immune system in mediating radiation nephropathy. This finding raised the untested possibility that T cells may also be responsible for cardiac remodeling. However, targeted irradiation of the kidneys with 10 Gy in T cell knock down Dahl S rats did not result in less fibrosis in the non-targeted heart. The high level of cardiac fibrosis in sham-irradiated wild-type and T cell knock down Dahl S rats likely obscured any additional effects of radiation [4]. To overcome this limitation, we adapted a model of genetic depletion of T cells to WAG rats, a strain of rat susceptible to non-targeted cardiac injury, to determine whether T cells are involved in communicating signals between the irradiated kidney and the non-targeted heart.

The objectives of our study were to (i) determine the timing and extent of immune cell infiltration in kidney and heart after targeted irradiation of kidneys (ii) generate and validate a T cell knock down rat on the WAG genetic background and (iii) determine the role of T cells on nephropathy caused by targeted irradiation of the kidneys as well as development of pathology in the non-targeted heart. The principal findings were that immune cell engagement following kidney irradiation is time dependent, and that T cells function as effector cells in communicating a signal from the irradiated kidneys that causes pathologic remodeling of non-targeted heart.

4. DiscussionOur findings show that the engagement of immune cells is part of a late response in radiation-damaged kidneys. It seems to involve several subsets, including CD3+, CD56+, and CD68+ positive cells that accumulate in irradiated kidneys four months after exposure coinciding with organ pathology. There was no accumulation of CD3+, CD56+, CD68+ or CD20+ positive cells in the kidney 40 days after irradiation, a time when BUN levels are increasing, but before organ pathology manifest as fibrosis is present. Immune cell accumulation in kidneys is associated with pathological remodeling of the heart outside the radiation field, and in the absence of cardiac immune infiltration indicates that the non-targeted effect on heart might not be directly mediated through cell-to-cell interaction with immune cells. This implies a mechanism that links local radiation injury of kidney to pathology in the non-targeted heart, which would require the transmission of signals from irradiated kidneys to non-irradiated heart. The identity of the systemic signals from the irradiated kidneys remains unclear. The initial response to local radiation may be sensed through conserved damage-associated molecular patterns signaling pathways (DAMPS) that ultimately form a link to the activation of the adaptive arm of the immune system [14,15,16,17].Using a CRISPR CD247 gene knock down model in WAG rats, we showed that radiation nephropathy is decreased in the absence of T cells. The involvement of T cells in driving radiation nephropathy is in line with our previous observation in T cell knock down Dahl S rats [4]. The fact that this was seen even without the addition of radiation in that strain underscores a potential role for T cells in driving kidney disease, whether radiation driven or age-related if the genetic predisposition is present [4]. The decrease in natural killer cells and macrophages in sham-irradiated kidneys from 20 days to 120 days may reflect a developmental decline in immune cells [18]. In the present study, we focused on intra-parenchymal immunocytes rather than those in the circulation because the intraparenchymal immune cells are apt to have a closer relation to the mechanisms of parenchymal injury. This local kidney irradiation model avoids the need for bone marrow transplantation and the confounding effect of donor lymphocytes.Irradiation of the kidneys in T cell knock down WAGCD247-/- rats limited the build-up of perivascular cardiac collagen typically seen in kidney-irradiated wild-type WAG rats [4]. Increased perivascular collagen deposition was not eliminated from kidney-irradiated T cell knock downWAGCD247-/- rats, indicating that cardiac fibrosis is not totally mediated by T cells. Increased perivascular cardiac collagen content is a marker for cardiac fibrosis and a hallmark of radiation-induced heart damage as shown by us [2,4]. In our study using the T cell depleted Dahl S rat, elevated levels of perivascular cardiac collagen already present in sham-irradiated wild-type and T cell depleted Dahl S rats compared with wild-type WAG rats likely obscured any additional effects of radiation [4].Humans have evolved in an environment continuously exposed to very low levels of background radiation from galactic cosmic rays, terrestrial sources from radionuclides, and intermittent exposure to radiation from solar particle events [19]. Exposure to low levels of ionizing radiation has not resulted in the evolution of unique mechanisms to directly detect exposure. There is no receptor that is specifically engaged by ionizing radiation in mammalian systems to initiate a biological response. The innate immune system functions as the sentinel alerting the body to tissue damage from radiation. Immune cells derived from both lymphoid and myeloid lineages in addition to T cells may be involved in the mechanism underlying non-targeted radiogenic pathology.In the setting of non-targeted effects, the heart would receive radiation from out-of-field sources; external air scatter originating from the X-ray tube that is independent of distance between anatomical sites and internal low dose X-ray scatter through the rat tissues that is dependent on anatomical distance. Radiation dose will likely be an important determinant in the type and extent of the response as different cellular mechanisms seem to have different thresholds, e.g., micronuclei formation or Nrf2 activation at 20 mGy [20] versus ER stress and proteasome changes at 50 mGy [21] versus ATP release at 100–250 mGy [22,23]. However, direct irradiation of the heart with 10 Gy did not result in pathology [2]. Future studies are needed to define the radiation dose to the kidneys that results in transmission of signals to heart.

Targeted radiation of the tumor is used for cancer treatment. The findings of the current study indicate that the impact of radiation outside the targeted volume needs to be considered in terms of side effects and overall outcomes. Organ systems are interdependent, with signals communicated between each other through the circulation, central nervous and lymphatic systems to maintain homeostasis. Thus, the biological consequences of radiogenic damage inflicted on a localized tumor and the surrounding normal tissue can be communicated to the rest of the body. Our studies confirm that irradiation of a cancer below the diaphragm could cause pathology in the non-targeted heart.

The identity and location of the source within the irradiated volume that communicates signals from cells in injured organs to cells in other organ systems such as the heart is undefined. This non-targeted effect was first measured in 1938 [24], after irradiating the lower third of a rat with 20–40 Gy X-rays. The upper third of the body was shielded to prevent direct actions of radiation on the thymus and spleen. Irradiation of the lower third of the body caused atrophy in the non-targeted and shielded thymus and spleen. In these studies, scatter irradiation may have contributed to the effects observed. This effect was defined as abscopal in 1953 [25] as “at a distance from the irradiated volume but within the same organism”. The findings of the present study indicate that T cells in the immune system are one of the sources of the signals responsible for communicating a signal from the irradiated kidney to the shielded and non-targeted heart. Our previous findings showed that cytokines such as eotaxin, IL-2, IL-13 and IL-18, resulting from targeted irradiation of the kidneys, may transmit signals from irradiated kidneys to the non-targeted heart to initiate pathology [4]. Non-targeted effects represent a paradigm shift from the ‘‘DNA centric’’ view that it is only ionizing radiation that elicits biological effects and subsequent adverse health outcomes solely because of an energy deposition event in the irradiated tissue [3].Previous studies have shown that macrophages and natural killer cells are the main infiltrating immune cell types in irradiated mouse kidney [26] and irradiated rat kidney [4], respectively. They also represent the more radioresistant fraction of immune cells. Radiation tends to skew the immune balance towards myeloid cells and Tregs, although our model cannot discern which subset is most relevant for driving heart disease. Future studies can test if myeloid cell depletion in our model would break the link between danger-sensing resident innate cells and T cells.The heart functions primarily as a pump [27] with a secondary function as an endocrine organ [28]. Impairment of heart function can result in symptomatic disease [27]. Radiotherapy increases the occurrence of heart disease in survivors of childhood cancer [29]. These survivors are at risk for harmful cardiovascular outcomes as late as 30 years after fractionated cardiac irradiation with 15 Gy or higher. Direct irradiation of the heart during radiation therapy for breast cancer in adults increases the frequency of major coronary events by 7.4% per Gy with no apparent threshold [30]. The increase in risk for major coronary events is proportional to the dose to the heart, begins within a few years of exposure, and continues for at least 20 years. Understanding the pathogenesis of radiation-induced heart disease is also relevant to setting exposure limits for astronauts in the context of exposure to space radiation during exploratory missions. NASA has designated lifetime radiation exposure limits to minimize or prevent risks of degenerative changes in heart and vasculature. These dose restrictions for the heart are based on direct irradiation of the heart and adjacent arteries [31]. The findings of the current study and others reveal adverse non-targeted effects of radiation on cardiac function [2,32] and structure [4], suggesting radiogenic cardiac pathology can arise from non-cardiac sources in organs below the diaphragm as well as from direct cardiac irradiation. Thus, one or more organs below the diaphragm are the source of a signal that results in disease in the non-targeted heart.Limitations. The present studies did not show significant immune cell infiltration in kidney 20 days after irradiation, a time when there is cytokine release [4]. Thus, cytokines may have been released from other components of the immune system, as well as non-immune sources such as endothelial cells and fibroblasts during the early response to irradiation of kidneys. We do not know how to link early cytokine release with late pathology in the heart. Future immune profiling investigations are needed to determine the timing and extent of immune cell infiltration in kidney. Studies are needed to examine the role of the large numbers of natural killer cells present in the kidney following irradiation, and in the mechanisms underlying the subsequent cardiac pathology. Our use of a constitutive gene knock down rat allowed us to show T cells play a major role in driving pathology to the non-targeted heart but did not completely explain this effect. This new finding provides the rationale to develop a time-dependent conditional gene knock down rat in which expression of the CD247 gene is temporally controlled to assess mechanisms. Our study focused on normal tissue injury. Further studies are needed to examine whether local irradiation of tumors results in pathology of non-targeted normal tissues such as heart.

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