Iron homeostasis plays a crucial role in the host's ability to resist infection by GBS.
In order to investigate whether ferroptosis occurs in the host following GBS infection, the survival rate of infected mice was assessed through the administration of the ferroptosis inhibitor Fer-1. In this study, the survival rate of treated mice was less than 50%, resulting in a significant decrease in overall survival rates (Fig. 1A). Concurrently, samples of peritoneal lavage fluid and peripheral blood were collected from mice at various time points (Fig. 1B, C). Analysis revealed that bacterial counts were significantly elevated in the GBS + Fer-1 group compared to the GBS infection group at 3, 6, and 15 hours post-infection, reaching a peak after 3 hours of treatment (Fig. 1B, C). These preliminarily results indicated that ferroptosis may be beneficial for the bacterial clearance in GBS infected mice. During the host immune response, the oxidative stress is commonly induced to kill invading bacteria, while biochemical effects such as iron, ROS, and GSH also are responsible for substantial changes. We assessed the peritoneal lipid peroxidation using liperfluoro (Dojindo, Japan), and the Flow cytometry analysis proved that mice infected with GBS showed a lipid peroxidation, while after treatment with fer-1, the lipid peroxidation was lower than that in the GBS infected group (Fig. 1D, E). The expression of PTGS2, a positive molecular marker of ferroptosis[15; 16], were significantly increased after 3 hours of infection from the peripheral lavage fluid, indicating that ferroptosis occurs in the host after GBS infection, playing a positive role in the resolution of the inflammation(Fig. 1F).
Additionally, we seek to investigate whether enhancing host resistance to GBS infection can be achieved through preemptive and sustained induction of ferroptosis. Based on this, we used Erastin, which is a recognized stimulator of ferroptosis[13], however, after the Erastin treatment of mice, we found that mice soccombed after GBS infection (Supplementary Fig. 1A), suggesting that a controled level of ferroptosis in the early stage of infection may play a crucial role in overcoming the infection, contrarily to a persistent or excessive ferroptosis that can weaken these effects.
Subsequently, in order to excluded the effects of other types of cell death on the immune response against GBS infection, we also evaluated the inhibitors 3-MA (3-methyladenine) and Nec-1 (Necrostatin-1), and found that they could also significantly inhibit the host resistance to GBS infection (Supplementary Fig. 1B). The relationship between autophagy and ferroptosis was extensively reported in the past[17; 18] and studies conducted on the correlation between Nec-1 and lipid oxidation showed that Nec-1 can inhibit ferroptosis caused by sulfasalazine and Erastin (system xc- inhibitors) and Rsl3 (a glutathione peroxidase 4 inhibitor)[19; 20]. In order to illustrate the inhibitory effect of Nec-1 here, we evaluated its effect on lipid oxidation after finding the ferroptosis stimulus. The results showed that Nec-1 could significantly inhibit lipid oxidation of macrophages. It suggested that the function of Nec-1 here is at least related to its inhibition of lipid oxidation to some extent (Supplementary Fig. 1C).
As Slc7a11 plays a crucial role in the system Xc−-GPX4-GSH pathway, we generated knockout mice to investigate the specific pathway implicated in lipid oxidation within the cell membrane. Nevertheless, the findings from repeated experiments indicated that the deletion of Slc7a11 resulted in a decrease in the host's capacity to combat GBS, although this difference was not deemed statistically significant (Supplementary Fig. 1D). The changes in the number of immune cells showed that only peritoneal macrophages significantly decreased in Slc7a11−/− mice after infection (Supplementary Fig. 1E). Based on the above results, lipid oxidation is indeed very important for the host to resist GBS, but Slc7a11 is not the main cause of lipid oxidation during GBS infection (Supplementary Fig. 1F,G). Meanwhile, there may be other mechanisms by which Nec-1 inhibits lipid oxidation which worth further study.
The ferroptosis of macrophages is important for the host response to GBS infection
The preliminary bacterial load and lipid oxidation results showed significant changes after 3 hours of infection, and we speculated that immune cells may also undergo significant changes during this time. After analyzing the peritoneal lavage fluid of infected mice, we found that the number of macrophages and neutrophils were significantly decreased after the inhibition of ferroptosis (Fig. 2A,B), indicating that the inhibition of ferroptosis reduce the host's ability to overcome the infection. Macrophages are widely distributed in peripheral tissues, which can destroy foreign pathogens through phagocytosis and play an indispensable role in defending pathogens. Among them, M1 cells exhibit effective antibacterial properties, such as release of pro-inflammatory cytokines and stimulation of the Th1 response, while M2 cells support Th2-related effector functions [7; 21]. Through flow cytometry analysis, we found that the changes of M1 macrophages were the most obvious. Indeed, once the ferroptosis was inhibited, the proportion of M1 macrophages significantly decreased, while M2 macrophages significantly increased(Fig. 2C,D). Therefore, we investigated if macrophages were the main cell type responsible for ferroptosis. Using clodronate liposome to block macrophages in GBS infected mice, we found that macrophage blockade significantly increased the mortality rate of infected mice (Fig. 2E). This rate was similar to the one observed in the Fer-1 treatment group, indicating that ferroptosis of macrophages plays an important role in the response to the infection. Subsequently, we investigated whether macrophages showed ferroptosis after infection. Peritoneal macrophages were isolated from mice at 3 hours after infection and were incubated with BODIPY 581/591 C11 for 0.5 hours to detect cellular lipid oxidation, which was observed within the cells after GBS infection (Fig. 2F). Previous literature reported that iron plays a crucial role in the immune response to bacterial infections, being also a key nutrient for bacterial survival in host cells[13]. We then investigated the intracellular ferrous content using FerroOrange incubation probe, and confocal imaging showed that after GBS infection, the intracellular ferrous fluorescence intensity elevated which indicated ferrous content increased (Fig. 2G), consistently with a significant increase in PTGS2 mRNA by qPCR (Fig. 2H). All the above results showed that the ferroptosis of macrophages is crucial for the host to develop an efficient response to GBS infection.
IL-1β may be the main reason for ferroptosis macrophages.
Since ferroptosis is important for host resistance to GBS infection, we then investigated the stimuli that cause ferroptosis in the host. Multiple previous studies showed that IL-1β can promote host defense against GBS infection by recruiting immune cells to the infected site. In this regard, Luminex results detected IL-1β 3 hours after GBS infection, with IL-6, IL-4, and IL-10 significantly increased, however, IL-1β and IL-6 decreased significantly after treatment with Fer-1, while anti-inflammatory factors IL-4 and IL-10 were significantly increased (Fig. 3A-E). At the same time, a significant increase in the number of bacteria was observed following the treatment with Fer-1 (Fig. 1B, C).It is suggested that the occurrence of ferroptosis in the early stage of infection can lead to the IL-1β upregulation.
Similar to Fer-1 treatment, the survival rate of mice in the IL-1β antibody blockade group was significantly reduced (Fig. 3F). Furthermore, we injected IL-1β intraperitoneally into mice, and it was found that IL-1β could significantly promote host resistance to GBS infection (Fig. 3G). Meanwhile, following the IL-1β antibody blockade, the degree of lipid oxidation in peritoneal macrophages was found to be significantly decreased during GBS infection, but slightly higher than the background (Fig. 3H). On the other hand, when we inject IL-1β (i.p.), we found that in the absence of GBS infection, peritoneal macrophages showed a ferroptosis characterized by increased lipid oxidation(Fig. 3I), further indicating that IL-1β may be the main stimulus for macrophage ferroptosis.
Afterwards, different concentrations of IL-1β were used to stimulate RAW264.7, and we found that 100pg/ml of IL-1β was able to significantly induce lipid oxidation in RAW264.7 (Fig. 4A, B), with a significant increase in RAW264.7 lipid oxidation at the timepoint of 0.5 hours (Fig. 4C). IL-1β has been identified as a potential inducer of ferroptosis in macrophages; however, findings from previous luminex assays suggest that the production of IL-1beta is also dependent on ferroptosis. Multiple studies also demonstrated a link between bacterial infection and ferroptosis in host cells, with the result to decreased the host's resistance to bacteria[22; 23; 24]. IL-1β is mainly produced by different type of cells during GBS infection, such as blood monocytes, tissue macrophages, and dendritic cells, while fibroblasts and epithelial cells usually are not involved in the production of cytokines[25].Therefore, we investigated whether GBS infection can directly cause ferroptosis in macrophage. Interestingly, our results showed a significant induction of lipid oxidation and expression of PTGS2 in RAW264.7 cells at 0.5 hours after infection when the MOI of GBS and RAW264.7 reached 10:1 or 30:1, while qPCR results detected IL-1β at a significant higher concentration, explaining why after IL-1β was blocked a significant level of ferroptosis was still observed in peritoneal macrophages(Supplementary Fig. 2A,B).Previous studies demonstrated that bacteria-induced ferroptosis is associated with an increase in intracellular ions[26], however, in the current study, GBS was not significantly associated with an increase in intracellular iron content by RAW 264.7 (Supplementary Fig. 2C). This suggested that GBS might directly causes ferroptosis of macrophages or some other cells in the early stages of infection, which promotes IL-1β production. As an inflammatory amplifier, IL-1β significantly activates the host's innate immune system and promotes the host's resistance to GBS infection.
IL-1β causes macrophage ferroptosis by directly causing lipid oxidation
As first line of defense against bacterial infections, macrophages developed several strategies for bacteria, such as limiting the iron acquisition, which is one of the most important regulatory pahway of host cells for iron homeostasis[27]. Considering the dependence of pathogenic microorganisms on iron, and it has been reported that IL-1β has a close relationship with iron transport by affecting the expression of hepcidin,FPN and CP[3; 28]. Is it thus likely that the excessive uptake of iron by cells is responsible for the ferroptosis caused by macrophage[29]. However, several experiments showed that, despite the constant observation of lipid oxidation in macrophages as well as the expression of PTGS2, there was no significant difference in the intracellular iron content between the infection and the control groups (Fig. 4D-F). Consistently, no significant changes were observed in the expression of key genes related to the metabolism of iron ions, such as FTH, FTL and FPN (Fig. 4G). But the qPCR results showed significantly increased levels of SLC7A11, GPX4, ALOX15, and LPCAT3 after IL-1β treatment (Fig. 5A). And the four genes aforementioned are closely related to lipid oxidation[30]. In detail, Slc7a11 is a key component of the system Xc, and the Xc-GSH-GPX4 axis is crucial for the cellular antioxidant activity, while ACSL4 and ALOX15 are key genes inducing phospholipid oxidation and they can be both inhibited by GPX4[30; 31; 32]. Therefore, we speculate a direct role of IL-1β in the lipid oxidation. Indeed, after inhibiting the expression of ALOX15 with inhibitor PD146176 [33], IL-1β was unable to cause lipid oxidation in macrophages anymore (Fig. 5B, C). Therefore, we infer that IL-1β may lead to ferroptosis in macrophages by directly inducing lipid oxidation.