Sub-lethal irradiation reduces red blood cells, hematocrit, and hemoglobin
Female C57Bl/6J mice were exposed to 6.85 Gy TBI (0.6 Gy/min). This sublethal dose of radiation did not result in mortality in any groups. Previous studies showed that radiation induces the hemolysis of RBC and reticulocytes due to the denaturation of hemoglobin 8,10. 6.85 Gy irradiation caused a significant reduction in RBCs, HCT, and HGB, with maximal ~60% reduction around day 14 (Fig. 1A-C). Because this sublethal radiation exposure dose causes only a relatively low level of blood cell reduction, no significant differences were observed between vehicle- and captopril-treated animals (Fig. 1A-C). The mean corpuscular volume (MCV), an indicator of the average erythrocyte size, was near baseline for days 7 and 14 post-irradiation in both vehicle and captopril groups (Fig. 1D). MCV exceeded the normal volumes on day 21 in vehicle-treated animals, but not in the captopril-treated group (Fig. 1D). Interestingly, the mean corpuscular HGB concentrations (MCHC) were maintained in captopril-treated mice over the full duration of the time course, while in vehicle-treated animals, the MCHC fell below basal levels at day 21 post-irradiation (Fig. 1E). Together, these data show that in the vehicle-treated group there is increased level of reticulocytes as indicated by the higher MCV along with corresponding lower mean concentration of hemoglobin per cell volume at 21 days. This represents an erythropoietic response to the loss of erythrocytes, with the loss most pronounced at 14-days. The increase in the circulating reticulocytes is further supported by the decreased MCHC in an inverse fashion compared to the change in the MCV. However, this increase is minimal in the case of captopril-treated group that has more gradual erythropoietic recovery. Overall there is an erythropoietic response most noticeable on day 21 for vehicle-treated group as indicated by the maximum MCV reflecting the robust release of immature erythrocytes from the bone marrow in the forms of larger reticulocytes to compensate the radiation-induced loss of erythrocytes.
Sub-lethal irradiation results in iron deposition in the spleen and upregulation of genes encoding iron handling proteins
Our laboratory and others demonstrated that following radiation-induced hemolysis, iron is deposited within the bone marrow 27,28. Iron recycling normally occurs within the spleen, where specialized macrophages take up senescent or damaged RBC 24. We therefore investigated spleen iron levels following TBI. Within 7-14 days, Prussian blue staining, that detects Fe3+, increased ~20-60-fold (Fig. 2A, B). Prussian blue staining returned to near basal levels by day 21. Histological analysis revealed that granular staining was primarily cytoplasmic within splenic histiocytes/macrophages. Captopril treatment did not significantly affect iron deposition in the spleen (Fig 2B).
We examined gene expression of iron binding and transport proteins following exposure to TBI (Fig. 3). Three patterns of gene expression changes were evident in the data. In the first group, Fth1 (ferritin heavy chain), Slc40a1 (ferroportin), and Trf (transferrin) were significantly upregulated at 7 days, with declining expression to near basal levels at 14-28 days post-irradiation (Fig. 3A-C). Ferritin heavy chain is associated with storage, primarily within macrophages. Iron is exported by cells through the transporter ferroportin that passes iron to transferrin, the primary protein for secure transport of iron through the plasma 23. The second group, Tfrc (CD71/ transferrin receptor), Itgam (integrin alphaM, also known as Mac-1 or CD11b/CD18), and Lcn2 (lipocalin-2) exhibited initial suppression followed by increased expression ~14-21 days (Fig. 3D-F). The CD71/transferrin receptor binds transferrin from the plasma for import of iron into the cell, while integrin alphaM has been shown to bind to and import iron oxide nanoparticles 29,30. Lipocalin-2 binds iron for uptake into cells where it can be sequestered 31-33. Finally, we examined expression of Flvcr1 which encodes two heme export proteins important for cellular heme homeostasis: feline leukemia virus subgroup C receptor 1a (FLVCR1a) a plasma membrane heme exporter, and FLVCR1b, a mitochondrial protein 34,35. Flvcr1 displayed reduced expression (~50%) at 28 days in vehicle-treated animals (Fig. 3G), suggesting a reduction in the uptake of heme-bound iron. Captopril treatment did not alter the expression of most of these genes. Captopril did enhance the expression of integrin alphaM at 21 days, with a trend toward increased expression at 28 days. Captopril also significantly enhanced the expression of lipocalin-2 at 21 and 28 days (Fig. 3E, F).
Immunohistochemistry (IHC) was used to identify cell types expressing several of the iron handling genes. As described above, ferritin gene expression increased ~7 days (Fig 4A, B). IHC showed a trend toward increased ferritin expression in the spleen at 7 days, declining at 14 and 21 days. Ferritin staining in irradiated spleens was granular in appearance and localized to the cytoplasm and dendritic processes of histiocytes/macrophages, with especially strong staining in cells in the sinuses under the spleen capsule (Fig. 4A). Western blotting showed significant increases in ferritin (~5-6-fold, p<0.05) at 7-14 days (Fig. 4C). Hemosiderin, an iron storage complex containing ferritin and Fe3+, appeared as gold-brown coloration (Fig. 4D). Data show that there was a significant increase in hemosiderin at 7-14 days (~4-fold higher than control, p<0.05), and 28 days (~2-fold higher than control, p<0.05).
qPCR showed an increase in the CD71/transferrin receptor, ~14-21 days post-irradiation. IHC showed a trend toward increased CD71 levels (~2-fold) 21 days post-irradiation (Fig. 5A, B). CD71 staining was prominent in red pulp, localized to the cytoplasmic membranes of erythrocytes, including nucleated immature erythrocytes. Staining was not identified within histiocytes/macrophages. Western blotting confirmed ~40-fold upregulation of ferritin at 7-14 days (p<0.05) (Fig. 5C). IHC was used to examine levels of CD163, a high affinity scavenger receptor for the hemoglobin-haptoglobin complex 36(Fig. 6A, B). CD163 levels were suppressed at all time points, to ~1/3 control levels. Staining was mostly present in the control samples, localized to the cytoplasm of histiocytes/macrophages of in the red pulp in the same cells that were stained with Prussian blue.
Reduction in spleen volume post-irradiation is associated with ferroptosis.
Our laboratory previously showed that the size of the spleen is reduced ~10 days following TBI in mice 37. We investigated the effect of radiation on the spleen weight, normalized to total body weight (Fig. 7A). The normalized spleen mass is reduced to ~50% of control (p<0.05) at 7 and 14 days post-irradiation in both vehicle and captopril treated animals, correlating with the times of highest levels of iron deposition in the spleen. Western blotting for upregulation of activated caspase 3 as a marker of programmed cell death revealed a significant increase in proteolytic fragments at 7 and 14 days post-irradiation in vehicle-treated mice (Fig. 7B, left panel; p<0.05). In captopril-treated animals, caspase 3 activation was also increased, significant at 14 days post-irradiation (Fig. 7B, right panel; p<0.05) Interestingly, we did not observe increased expression of p21/Waf1, a marker of cell cycle inhibition and accelerated senescence (Fig. S1).
Recent studies showed that high levels of iron can induce ferroptosis, a form of programmed cell death 38,39. Ferroptosis markers include increased expression of CD71/ferritin receptor 1, and reduced glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (Slc7A11) 38,40. We demonstrated increased expression of the CD71 using qPCR (Fig. 3) and western blotting (Fig. 5B); IHC of the spleen showed that this increase occurred within mature and immature erythrocytes. We next investigated the gene expression of Gpx4 and Slc7a11 in the total spleen tissue (Fig. 7C,D). Gpx4 expression showed a trend toward decreased expression (~25% reduced) within 7 days post-irradiation, and was significantly suppressed, to less than 50% basal levels, at 14 and 21 days post-irradiation (p<0.001-0.0001) compared with basal levels. In vehicle-treated animals Gpx4 returned to near basal levels at 28 days post-irradiation; captopril treatment resulted in Gpx4 remaining significantly lower than basal levels at 28 days (p<0.05). In contrast, Slc7a11 showed a slight trend toward increased expression at 7 days post-irradiation, followed by a trend toward reduced expression at 14 days post-irradiation in both vehicle- and captopril-treated groups. Slc7a11 was significantly reduced only in vehicle-treated animals at 21 days post-irradiation (p<0.0001). In captopril-treated animals, Slc7a11 remained near basal levels over the time course.
Cyclooxygenase-2 (COX-2) is also marker, but not a driver, of ferroptosis 38. Western blotting showed that COX-2 protein levels were significantly increased at 7 days post-irradiation in vehicle-treated animals (Fig. 7E, left panel; p<0.05). We observed a trend toward increased COX-2 protein in captopril-treated animals, but this did not reach significance (Fig. 7E, right panel).
Iron- and radiation-induced alterations in gene expression in culture murine macrophages.
Macrophages play a key role in iron homeostasis as well as in normal immune responses 23. Iron regulates iron binding and transport proteins in macrophages and modulates macrophage polarity 17,41,42. Iron exposure upregulates ferritin and several other iron storage and transport proteins in macrophages 17, and impairs the ability of macrophages to assume a full pro-inflammatory (M1) phenotype 41. However, in vitro studies showed that radiation had no effects on macrophage polarity 43. In contrast with in vitro studies, in vivo studies of TBI have shown effects on macrophage polarization, favoring the induction of M1 polarization early after radiation, followed by the development of alternatively activated (anti-inflammatory or M2) macrophages 44,45.
Because the in vivo effects of total body radiation likely reflect the impact of radiation with iron on macrophages, we wished to determine the effects of iron, radiation, and iron + radiation on macrophage polarization in vitro. We utilized 7 and 12.5 mg/L Fe3+ in the medium based on reported iron concentrations in the serum following TBI in mice 10; iron was combined with two doses of radiation that are sublethal when used in TBI studies in C57BL/6 mice 46. We examined the gene expression of three markers of pro-inflammatory M1 polarization, inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and IL-1β (Fig. 8A-C). Of these, radiation significantly induced Nos2 and Il1b, but not Il6. Iron alone did not activate any markers of M1 polarity, but the presence of iron in the medium did show a trend toward augmenting iNOS expression (Fig. 8A). Neither iron alone nor radiation alone activated the M2 marker Arg1 (arginase 1) (Fig. 8D). Iron + radiation did result in the upregulation of Arg1, although not consistent for all radiation + iron conditions (Fig. 8D). We next examined the ability of M1- or M2-inducing cytokines (IFN-γ + LPS for M1; IL-4 for M2) to induce polarization in the presence of iron, radiation, or iron + radiation (Fig. 9). Iron, radiation, or iron + radiation did not inhibit the upregulation of Il6 or Il1b induction by IFN-γ + LPS or Arg1 induction by IL-4. Together, these data suggest that radiation and iron + radiation may induce mixed polarity but they do completely block cytokine responses by the macrophages.
Our in vivo data indicated that macrophages in the spleen contain high levels of ferritin after TBI. To determine the effects of high iron concentrations and ionizing radiation on iron binding protein expression, we examined the levels ferritin in J774A.1 cultured macrophages. We found ~6-14 fold upregulation of ferritin heavy chain in all cultures that included high iron within 24 h (p<0.05, Fig. 10A). Interestingly, upregulation of ferritin heavy chain protein was not significantly affected by radiation exposure or by M1- or M2-inducing cytokines, although radiation and M1-inducing cytokines did upregulate gene expression of Fth at 24 h (Fig. S2). We did not detect significant levels of transferrin receptor 1 in the cultured macrophages before or after irradiation.
In vivo data also indicated that significant levels of programmed cell death, but not accelerated senescence, occurred in the spleen following TBI. However, our previous studies indicated that ionizing radiation induces accelerated senescence in cultured pulmonary artery endothelial cells 47. The spleen contains significant levels of macrophages as well as microvascular endothelial cells. We investigated programmed cell death and senescence in cultured J774A.1 and human spleen microvascular endothelial cells. J774A.1 cells displayed a trend toward increase in p21/waf1, a marker for accelerated senescence, after exposure to 2 or 6.85 Gy ionizing radiation (Fig. 10B). Senescence was significant in 6.85 Gy irradiated cells in the presence of iron (Fig. 10B). Neither iron alone nor M1- or M2-inducing cytokines induced p21/waf1. We did not detect significant caspase-3 activation under any conditions (data not shown). Because the spleen contains a fairly large population of microvascular endothelial cells, we investigated the effects of radiation and high iron exposure on human spleen microvascular endothelial cells (HSpMVEC) in culture. Interestingly, we were unable to detect either caspase-3 activation or p21/waf1 upregulation in HSpMVEC at 24 h following radiation, iron, or iron + radiation exposure (Fig. S3).