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Cellular iron governs the host response to malaria [1]

['Sarah K. Wideman', 'Mrc Human Immunology Unit', 'Mrc Weatherall Institute Of Molecular Medicine', 'University Of Oxford', 'John Radcliffe Hospital', 'Oxford', 'United Kingdom', 'Joe N. Frost', 'Felix C. Richter', 'Kennedy Institute Of Rheumatology']

Date: 2023-10

Malaria and iron deficiency are major global health problems with extensive epidemiological overlap. Iron deficiency-induced anaemia can protect the host from malaria by limiting parasite growth. On the other hand, iron deficiency can significantly disrupt immune cell function. However, the impact of host cell iron scarcity beyond anaemia remains elusive in malaria. To address this, we employed a transgenic mouse model carrying a mutation in the transferrin receptor (Tfrc Y20H/Y20H ), which limits the ability of cells to internalise iron from plasma. At homeostasis Tfrc Y20H/Y20H mice appear healthy and are not anaemic. However, Tfrc Y20H/Y20H mice infected with Plasmodium chabaudi chabaudi AS showed significantly higher peak parasitaemia and body weight loss. We found that Tfrc Y20H/Y20H mice displayed a similar trajectory of malaria-induced anaemia as wild-type mice, and elevated circulating iron did not increase peak parasitaemia. Instead, P. chabaudi infected Tfrc Y20H/Y20H mice had an impaired innate and adaptive immune response, marked by decreased cell proliferation and cytokine production. Moreover, we demonstrated that these immune cell impairments were cell-intrinsic, as ex vivo iron supplementation fully recovered CD4 + T cell and B cell function. Despite the inhibited immune response and increased parasitaemia, Tfrc Y20H/Y20H mice displayed mitigated liver damage, characterised by decreased parasite sequestration in the liver and an attenuated hepatic immune response. Together, these results show that host cell iron scarcity inhibits the immune response but prevents excessive hepatic tissue damage during malaria infection. These divergent effects shed light on the role of iron in the complex balance between protection and pathology in malaria.

Malaria is a serious and potentially lethal infectious disease that affects nearly 250 million people each year. It is caused by Plasmodium species parasites that are transmitted between humans by mosquitoes. Iron deficiency is prevalent in malaria endemic areas and there is a complex and incompletely understood relationship between iron and malaria. Although iron deficiency is known be protective in malaria, little is known about how iron deficiency affects host cells other than the red blood cells where Plasmodium replicates, such as immune, liver, lung or kidney cells. To address this, we used genetically modified mice with decreased cellular iron uptake, but no anaemia, and infected them with a mouse strain of malaria. These mice had a more severe infection, characterised by more infected red blood cells and more weight loss at the peak of infection compared to wild-type mice. Interestingly, the mice had a significantly weaker immune response but also less severe liver damage upon malaria infection, indicating a trade-off between pathogen control and host health. This study highlights the key role of host iron status in malaria and may have implications for the treatment approach to both malaria and iron deficiency in malaria endemic regions.

Funding: This work was supported by a Wellcome Trust Infection, Immunology & Translational Medicine doctoral programme grant awarded to SKW, grant no. 108869/Z/15/Z, UK Medical Research Council MRC Human Immunology Unit core funding awarded to HD, grant no. MC_UU_12010/10 (JNF, CN, AEP, NW, SY, AEA, HD), the Clarendon Fund and the Corpus Christi College A. E. Haigh graduate scholarship (WRT), Portuguese National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020 (JML, NV, TLD), a Wellcome Trust Infection, Immunology & Translational Medicine doctoral programme grant (awarded to FCR, grant no. 203803/Z16/Z), and a Wellcome Trust Senior Fellowship (106917/Z/15/Z, SJD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AEA and HD received salaries from MRC UK; SKW, FCR and SJD received salaries or salary contributions from Wellcome Trust; MRT received salary from the Clarendon Fund.

In this study, we aspired to deepen our understanding of how malaria infection is affected by host iron deficiency. To this end, we employed a genetic mouse model of cellular iron deficiency based on a rare mutation in TfR1 (Tfrc Y20H/Y20H ), which causes combined immunodeficiency in humans [ 29 , 30 ]. We found that decreasing host cellular iron levels increased peak malaria parasitaemia in mice infected with P. chabaudi. While P. chabaudi-induced anaemia and RBC invasion remained unaffected, the immune response to P. chabaudi was drastically inhibited. Interestingly, mice with cellular iron deficiency also had attenuated P. chabaudi-induced liver damage, suggesting reduced immunopathology. Hence, host cellular iron deficiency attenuated the immune response to malaria, leading to increased pathogen burden and mitigated liver pathology.

Although it is known that host iron deficiency influences malaria infection, the mechanisms that affect host health or Plasmodium virulence remain largely unknown. In particular, the effects of iron deficiency aside from anaemia, have scarcely been explored. Moreover, any effects on malaria immunity have not been investigated beyond a few observational studies that found associations between iron deficiency and attenuated antibody responses to malaria in children [ 7 , 44 , 45 ].

Controlling a malaria infection requires two distinct but complementary immune responses. An early cell-mediated response, primarily driven by interferon-γ (IFN-γ) producing CD4 + T cells, prevents uncontrolled exponential parasite growth [ 31 – 35 ]. Meanwhile, a humoral response is required to prevent recrudescence and to clear the infection [ 36 , 37 ]. Excessive production of pro-inflammatory immune cells and cytokines can lead to sepsis-like complications and cause collateral damage to tissues and organs [ 38 , 39 ]. Thus, the pro-inflammatory anti-parasite response must be balanced by immunoregulatory and tissue-protective responses to prevent immunopathology [ 40 – 43 ].

Anaemia is the primary and most profound consequence of iron deficiency. However, iron deficiency can also have other negative impacts on human health. Immune cells with high proliferative and anabolic capacities appear to be particularly sensitive to iron deficiency. As such, decreased iron availability can impair the proliferation and maturation of lymphocytes and neutrophils [ 14 – 16 ]. Neutrophils and macrophages also require iron for enzymes involved in microbial killing [ 16 – 19 ]. In animal models of iron deficiency, lymphocyte function is severely impaired, and the immune response to immunisation and viral infection is inhibited [ 20 , 21 ]. Similarly, iron deficiency decreases inflammation and improves outcomes in mouse models of autoimmune disease [ 22 – 25 ]. In humans, associations between iron deficiency and attenuated responses to some vaccines have been observed [ 20 , 21 , 26 – 28 ]. Moreover, patients with a rare mutation in transferrin receptor-1 (TfR1), the primary receptor for iron uptake in cells, present with lymphocyte dysfunction and combined immunodeficiency [ 29 , 30 ].

Meanwhile, oral iron supplementation is a risk factor for malaria in areas with limited access to preventative measures and treatment [ 11 , 12 ]. This effect can to some extent be explained by iron supplementation stimulating erythropoiesis and increasing the proportion of reticulocytes and young erythrocytes, which are preferred targets for invasion by P. falciparum parasites [ 10 ]. Malaria and iron deficiency also often disproportionally affect the same populations (e.g. young children in the WHO African Region) [ 1 , 6 ], in part, because malaria causes iron deficiency [ 13 ].

There is a complex relationship between host iron status and malaria. Iron is an essential micronutrient that is required by most living organisms to maintain physiological and biochemical processes, such as oxygen transport and storage, cellular metabolism, and reduction-oxidation reactions [ 3 , 4 ]. Despite the importance of iron, iron deficiency is exceedingly common in humans, and iron deficiency anaemia is estimated to affect a sixth of the world’s population [ 5 , 6 ]. In the context of human malaria infection, iron deficiency can decrease the risk of disease, severe disease, and mortality [ 7 – 9 ]. The protective effect of iron deficiency is at least partly mediated by anaemia, as RBCs isolated from anaemic individuals are less amenable to malaria parasite growth [ 10 ].

Malaria is a major global health problem that causes significant morbidity and mortality worldwide [ 1 ]. It is caused by Plasmodium species parasites, which have a complex life cycle and are transmitted between humans by Anopheles mosquitos. In the human host, multiple cycles of asexual parasite replication inside red blood cells (RBC) result in extensive RBC destruction, immune activation, and microvascular obstruction [ 2 ]. This blood stage of infection gives rise to symptoms such as fever, chills, headache, and malaise. In severe cases, it can also cause life-threatening complications such as acute anaemia, coma, respiratory distress, and organ failure [ 2 ].

Inflammation also causes severe disease and liver pathology in malaria [ 39 , 61 , 64 ]. Hence, hepatic inflammation was approximated by measuring the expression of genes encoding pro-inflammatory cytokines IFNγ, TNFα, and IL-1β. We observed no difference in the expression of Ifng or Tnf, but Il1b expression was lower in Tfrc Y20H/Y20H mice eight days after P. chabaudi infection ( S5 Fig ). Moreover, immunohistochemistry staining showed reduced infiltration of leukocytes (CD45 + cells) in livers of Tfrc Y20H/Y20H mice ( Fig 7G and 7H ). Additionally, a smaller proportion of liver leukocytes (CD45 + ) were effector immune cells such as dendritic cells, CD44 + CD4 + T cells, and CD44 + CD8 + T cells ( Fig 7I and 7L ). Taken together, this data shows that host cell iron scarcity leads to an attenuated hepatic immune response during P. chabaudi infection.

During malaria infection, endothelial activation leads to increased adhesion and sequestration of iRBCs, resulting in hepatic vascular occlusions and hypoxia that cause damage [ 2 , 63 ]. Fewer sequestration, rosetting, and vascular occlusion events were detected in liver sections from Tfrc Y20H/Y20H mice eight days after P. chabaudi infection ( Fig 7F ). Together with the trend toward lower ANG-2 levels in Tfrc Y20H/Y20H mice ( Fig 7A ), this indicates that decreased endothelial activation and iRBC sequestration contributed to the attenuated liver pathology observed in Tfrc Y20H/Y20H mice.

Excess reactive liver iron and haem are known to cause liver damage in malaria [ 61 , 62 ]. However, we observed no differences in total non-haem liver iron ( Fig 1I ) or liver lipid peroxidation, which correlates with ROS levels ( S5 Fig ). Hence, it is unlikely that tissue level variations in hepatic reactive iron or haem can explain the difference in liver damage. In addition, we measured the expression of two genes that are known to have a hepatoprotective effect in the context of iron loading in malaria: Hmox1 (encodes haemoxygenase-1 (HO-1)) and Fth1 (encodes ferritin heavy chain). Liver gene expression of Hmox1 was higher in Tfrc Y20H/Y20H mice, while the expression of Fth1 did not differ between genotypes, eight days after infection ( S5 Fig ). Thus, the higher expression of Hmox1 may have contributed to a hepatoprotective effect in Tfrc Y20H/Y20H mice.

Histological analysis revealed hepatic pathology in all P. chabaudi infected mice, characterised by hepatocellular necrosis, sinusoidal dilatation, glycogen depletion, and infiltration by mononuclear immune cells (Figs 7C, 7D and S5 ). Interestingly, no polymorphonuclear immune cell infiltration was observed. All infected wild-type mice developed confluent necrosis (areas of lobular disarray, eosinophilia, and loss of glycogen deposits, score ≥3), and most individuals (8 out of 11) also displayed bridging necrosis (areas of confluent necrosis extending across multiple lobules, score = 4) (Figs 7E and S5 ). In contrast, severe focal necrosis or confluent necrosis (score ≥3) was detected in just over half (6 out of 10) infected Tfrc Y20H/Y20H mice, and only four individuals developed bridging necrosis (Figs 7E and S5 ). Hence, the proportion of mice that developed severe hepatic necro-inflammation (score ≥3) upon P. chabaudi infection was significantly smaller in Tfrc Y20H/Y20H than in wild-type mice ( Fig 7E ).

Liver pathology of P. chabaudi infected C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice, 8 days after infection. A-B) Serum levels of angiopoietin-2 (A) and alanine transaminase (B). Mean, Welch’s t-test, n = 15–16. Dotted line represents uninfected mice. C-D) Haematoxylin and eosin (C), and periodic acid–Schiff (D) staining of representative liver sections. Labels indicate central veins (CV), portal triads (PT), and areas of focal (black arrows) and bridging (white arrows) necrosis. Original magnification 40X, scale bar 100 μm. E) Quantification of severe hepatic necrosis (score ≧ 3) as measured by histological scoring. Count, Fisher’s exact test, n = 10–11. F) Number of hepatic red blood cell sequestration, rosetting and vascular occlusion events per randomly imaged high-power field (HPF). Mean, Welch’s t-test, n = 10–11. G) Immunohistochemistry staining of liver leukocytes (CD45 + ) in representative liver sections. Original magnification 20X, scale bar 100 μm. H) Quantification of CD45 + leukocytes in liver sections identified by immunohistochemistry staining. n = 9–11 . I-L) Hepatic monocytes/macrophages (I), dendritic cells (J), CD44 + CD4 + T cells (K) and CD44 + CD8 + T cells (L). Mean, Welch’s t-test, n = 7–12.

We first measured circulating levels of angiopoietin-2 (ANG-2) and alanine transferase (ALT). ANG-2 is a marker of endothelial activation that correlates with malaria disease severity and mortality in humans [ 58 , 59 ]. Liver damage is also indicative of severe malaria [ 60 ], and ALT is a standard marker of liver damage. There was a trend towards lower ANG-2 and significantly decreased ALT in Tfrc Y20H/Y20H mice eight days after P. chabaudi infection, suggesting milder pathology ( Fig 7A and 7B ). Considering the substantial difference in serum ALT between genotypes, we further examined the malaria induced liver pathology. Tfrc Y20H/Y20H mice had lower expression of the tissue-damage and inflammation-induced acute phase protein genes Saa1 and Fga ( S5 Fig ). Furthermore, while both genotypes developed malaria-induced hepatomegaly, there was a trend toward less severe hepatomegaly in Tfrc Y20H/Y20H mice ( S5 Fig ).

Tfrc Y20H/Y20H mice experienced higher P. chabaudi parasitaemia and an inhibited immune response. However, the precise consequences of this disease phenotype remained unclear. Aspects of the immune response, such as the cytokine profile and the balance between pro-inflammatory and immunoregulatory responses, can tip the scales toward protection or pathology in malaria [ 39 ]. Hence, an attenuated immune response could cause hyperparasitaemia, but it may also be crucial in limiting immunopathology. We therefore set out to characterise key indicators of malaria disease severity.

A) B cells were isolated from uninfected C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice and cultured for 72 h in B cell activating media, with varying concentrations of ammonium ferric citrate (AFeC). B) Large neutral amino acid transporter-1 (LAT-1/CD98) expression on divided B cells, measured through geometric mean fluorescence intensity. Mean, two-way ANOVA, Sidak’s multiple comparisons test, n = 3. C) Representative flow cytometry plot of B cell proliferation, measured through CellTrace Violet (CTV) labelling. D) Proportion of proliferating B cells (CTV low ). Mean, two-way ANOVA, Sidak’s multiple comparisons test, n = 3. E) Representative flow cytometry plots of antibody secreting (CD138 + ) and class-switched (IgG + ) divided B cells. F-G) Proportion of antibody secreting (F) and class-switched (G) divided B cells. Mean, two-way ANOVA, Sidak’s multiple comparisons test, n = 3.

To determine if the Tfrc Y20H/Y20H mutation also had cell-intrinsic and iron-dependent effects on B cells, their functionality was further investigated in vitro. B cells were isolated from uninfected Tfrc Y20H/Y20H and wild-type mice, activated, and cultured in standard or iron-supplemented media for three days ( Fig 6A ). Expression of the B cell activation marker LAT-1 was lower on Tfrc Y20H/Y20H B cells than wild-type ( Fig 6B ). However, LAT-1 expression was rescued by iron supplementation, indicating improved B cell activation ( Fig 6B ). Tfrc Y20H/Y20H B cell proliferation was also severely impaired compared to wild-type cells, but was rescued by iron supplementation in a dose-dependent manner ( Fig 6C and 6D ). Iron scarcity also inhibited the potential of Tfrc Y20H/Y20H B cells to differentiate into antibody-secreting and class-switched cells ( Fig 6E, 6F and 6G ). This impairment was fully restored upon iron supplementation ( Fig 6E, 6F and 6G ). Overall, our data clearly show that the activation, proliferation, and differentiation of Tfrc Y20H/Y20H B cells were impaired, demonstrating that cellular iron deficiency causes cell-intrinsic B cell dysfunction.

We observed no difference between genotypes in the total number of splenic B cells at the acute stage of infection (8 dpi) ( Fig 5D ). However, mice with decreased cellular iron uptake had severely impaired B cell activation and fewer antibody-secreting effector B cells ( Fig 5E and 5F ). Additionally, Tfrc Y20H/Y20H mice had fewer GC B cells during acute infection (8 dpi) ( Fig 5G ). This effect remained in the chronic stage of infection (20 dpi) ( Fig 5H and 5I ), indicating a prolonged immune inhibition caused by restricted cellular iron availability.

Splenic immune response of P. chabaudi infected C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice. A) Proportion of CD4 + T cells expressing B cell co-stimulatory receptor ICOS, 8 days post infection (dpi). Mean, Welch’s t-test, n = 10–11. B) Proportion of T follicular helper (Tfh) cells, 8 dpi. Mean, Welch’s t-test, n = 9. C) Proportion of Tfh cells, 20 dpi. Mean, Welch’s t-test, n = 6–7. D-F) Total number of splenic B cells (D) and proportion of activated (E) and antibody secreting (F) splenic B cells, 8 dpi. Mean, Welch’s t-test, n = 9. G) Proportion of germinal centre B cells, 8 dpi. Mean, Welch’s t-test, n = 9. H) Representative flow cytometry plot of germinal centre B cells, 20 dpi. I) Proportion of germinal centre B cells, 20 dpi. Mean, Welch’s t-test on log transformed data, n = 6–9. Dotted line represents uninfected mice.

An efficient germinal centre (GC) response is required to generate high-affinity antibodies that enable malaria clearance [ 36 , 37 ]. In light of the impaired CD4 + T cell response to P. chabaudi in Tfrc Y20H/Y20H mice, we further examined the B cell supporting T follicular helper cell (Tfh) response. During the acute stage of infection, a smaller proportion of CD4 + T cells from Tfrc Y20H/Y20H mice expressed B cell co-stimulation receptor ICOS ( Fig 5A ). ICOS is essential in malaria infection, as it is required to maintain the Tfh cell response and sustain antibody production [ 57 ]. In line with this, Tfrc Y20H/Y20H mice had fewer Tfh cells, both during the acute (8 dpi) and chronic (20 dpi) stages of infection ( Fig 5B and 5C ). Tfh cells support the activation, differentiation, and selection of high-affinity GC B cells, and are an essential component of the humoral immune response to malaria [ 37 ]. Therefore, we next sought to assess the B cell response to P. chabaudi infection in Tfrc Y20H/Y20H and wild-type mice.

A) Naïve CD4 + T cells were isolated from uninfected C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice, and cultured for 96 h in Th1 polarising media, with varying concentrations of iron sulfate (FeSO 4 ). B) Representative flow cytometry plot of CD4 + T cell proliferation, quantified using CellTrace Violet. C) Proportion of CD4 + T cells that have divided more than two times (> 2X). Mean, two-way ANOVA, Sidak’s multiple comparisons test, n = 3. D) Representative flow cytometry plot of IFNγ producing CD4 + T cells in the absence or presence of FeSO 4 . E-F) Proportion of IFNγ producing CD4 + T cells (E) and IFNγ production per cell measured through geometric mean fluorescence intensity (gMFI) (F). Mean, two-way ANOVA, Sidak’s multiple comparisons test, n = 3.

To determine whether these impairments were T cell intrinsic and iron-dependent, we utilized naïve CD4 + T cells isolated from uninfected wild-type and Tfrc Y20H/Y20H mice. The cells were cultured in vitro under Th1 polarising conditions for four days, in standard or iron-supplemented culture media ( Fig 4A ). Tfrc Y20H/Y20H lymphocytes can acquire iron under conditions where transferrin is hyper-saturated and sufficient quantities of free iron are likely to be generated [ 29 , 56 ]. Proliferation was significantly impaired in Tfrc Y20H/Y20H CD4 + T cells but could be rescued in a dose-dependent manner by iron supplementation ( Fig 4B and 4C ). In addition, very few Tfrc Y20H/Y20H CD4 + T cells cultured in standard media produced IFNγ. However, iron supplementation completely rescued IFNγ production ( Fig 4D, 4E and 4F ). Hence, the CD4 + T cell deficiencies observed in Tfrc Y20H/Y20H mice during P. chabaudi infection were replicated in vitro and could be rescued by iron supplementation. These observations confirm that host cell iron scarcity disrupts CD4 + T cell function, leading to an inhibited CD4 + T cell response to P. chabaudi infection.

T helper 1 (Th1) cells and other T helper subsets that express IFNγ are particularly important for malaria immunity [ 55 ]. Interestingly, the proportion of CD4 + T cells that expressed the Th1 transcription factor T-BET was lower in mice with decreased cellular iron uptake ( Fig 3G ). Furthermore, fewer CD4 + T cells from Tfrc Y20H/Y20H mice produced IFNγ upon ex vivo restimulation ( Fig 3H and 3I ). Thus, further strengthening the evidence of functional CD4 + T cell impairment in Tfrc Y20H/Y20H mice during P. chabaudi infection.

Similarly, the total CD8 + T cell count did not differ between genotypes ( S4 Fig ), but P. chabaudi infected Tfrc Y20H/Y20H mice had fewer effector CD8 + T cells eight days after infection ( S4 Fig ). However, there was no difference in the percentage of antigen-experienced (CD44 + or PD-1 + ) ( S4 Fig ), proliferating (KI-67 + ) ( S4 Fig ) or IFNγ producing ( S4 Fig ) CD8 + T cells. Hence the CD8 + T cell response to P. chabaudi infection was also attenuated, albeit to a lesser degree than CD4 + T cells.

Conventional CD4 + T cells (FOXP3 - ) in the spleen of P. chabaudi infected C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice, 8 days after infection. A) Absolute number of CD4 + T cells. Mean, Welch’s t-test, n = 9–11. B) Proportions of naïve (CD44 - CD62L + ), effector (CD62L - CD127 - ) and memory (CD44 + CD127 + ) CD4 + T cells. Mean, two-way ANOVA with Sidak’s multiple comparisons test, n = 9–11. C) Absolute number of effector CD4 + T cells. Mean, Mann-Whitney test, n = 9–11. D-E) Proportions of CD4 + T cells expressing markers of antigen experience CD44 + (D) and PD-1 + (E). Mean, Welch’s t-test n = 9–11. F) Proportion of proliferating (KI-67 + ) CD4 + T cells. Mean, Welch’s t-test n = 9–11. G) Proportion of T helper 1 (TBET + ) CD4 + T cells. Mean, Welch’s t-test n = 9–11 . H) Representative flow cytometry plot of IFNγ producing CD4 + T cells, detected by intracellular cytokine staining. I) Proportion of IFNγ producing CD4 + T cells. Mean, Welch’s t-test n = 10–11. Dotted line represents uninfected mice.

T cells, particularly CD4 + T cells, are a critical component of the immune response to blood-stage malaria [ 55 ]. Therefore, we assessed the splenic T cell response to acute P. chabaudi infection. The total splenic CD4 + T cell count was comparable in both genotypes eight days after infection ( Fig 3A ). However, mice with decreased cellular iron uptake had a decreased proportion of effector CD4 + T cells ( Fig 3B ), and, consequently, fewer total splenic effector CD4 + T cells than wild-type mice ( Fig 3C ). In addition, the proportion of antigen-experienced CD44 + and PD1 + CD4 + T cells was also reduced in Tfrc Y20H/Y20H mice, re-enforcing their less activated state ( Fig 3D and 3E ). Moreover, fewer Tfrc Y20H/Y20H CD4 + T cells were actively dividing, based on the proliferation marker KI-67 ( Fig 3F ). This suggests a functional impairment of the CD4 + T cell response to P. chabaudi in mice with decreased cellular iron uptake.

Malaria infection leads to an influx of mononuclear phagocytes (MNP) into the spleen, where they are involved in cytokine production, antigen presentation, and phagocytosis of iRBCs [ 34 , 35 , 43 ]. Upon P. chabaudi infection, fewer MNPs were detected in the spleen of Tfrc Y20H/Y20H mice ( Fig 2C ). This applied both to CD11b + Ly6C + MNPs (resembling inflammatory monocytes and/or monocyte-derived macrophages) and to CD11c + MHCII + MNPs (resembling dendritic cells) (Figs 2D, 2E and S3 ). In malaria infection, some MNPs can produce IFNγ that facilitates naïve CD4 + T cell activation and polarisation [ 34 ]. Consequently, splenocytes from infected mice were cultured ex vivo with a protein transport inhibitor, and intracellular cytokine staining was performed. Interestingly, fewer MNPs from Tfrc Y20H/Y20H mice produced IFNγ compared to MNPs from wild-type mice (Figs 2F and 2G ). Infected wild-type and Tfrc Y20H/Y20H mice had comparable splenic neutrophil, eosinophil and NK cell numbers during acute infection (8 dpi) ( S3 Fig ). Thus, mice with decreased cellular iron uptake had an attenuated MNP response to P. chabaudi infection.

Splenic immune response to P.chabaudi in C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice at 8 days after infection. A) Representative picture of spleens from naïve and P. chabaudi infected mice. B) Spleen index of spleens from P. chabaudi infected mice. Mean, Welch’s t-test n = 9. C-E) Absolute numbers of CD11b + CD11c + mononuclear phagocytes (MNPs) (C), Ly6C hi CD11b hi monocytes/macrophages (D) and MHCII + CD11c + dendritic cells (E). Mean, Welch’s t-test on untransformed (C) or log transformed data (D, E) n = 9–11. F) Representative flow cytometry plot of interferon-γ (IFNγ) production of CD11b + CD11c + MNPs. G) Proportion of IFNγ-producing MNPs, detected by intracellular cytokine staining. Mean, Welch’s t-test n = 9–11. Dotted line represents uninfected mice.

The immune response to malaria exerts control of parasitaemia, and the spleen is the main site of the immune response to blood-stage malaria [ 39 , 54 ]. Therefore, we assessed the splenic immune response to P. chabaudi during the acute stage of infection (8 dpi). Interestingly, Tfrc Y20H/Y20H mice had attenuated splenomegaly during acute P. chabaudi infection ( Fig 2A and 2B ), suggesting a disrupted splenic response.

The elevated serum iron observed in infected Tfrc Y20H/Y20H mice was consistent with their restricted capacity to take up circulating transferrin-bound iron into tissues. However, we decided to investigate whether this supraphysiological serum iron (i.e., hyperferremia) could alter P. chabaudi parasite growth. To do this, we treated wild-type mice with a recombinant monoclonal anti-BMP6 IgG antibody (αBMP6) or an isotype control ( S2 Fig ). αBMP6 treatment suppresses hepcidin expression and elevates serum iron, as a consequence of unregulated release of iron from cellular stores [ 53 ] ( S2 Fig ). P. chabaudi infected mice treated with αBMP6 had higher serum iron than isotype control-treated mice on days 9 and 21 after infection ( S2 Fig ). Nevertheless, mice treated with αBMP6 and isotype had comparable peak parasitaemia and peak iRBC counts, although αBMP6 treated mice appeared to clear the parasites slightly more efficiently ( S2 Fig ). In addition, αBMP6 treatment did not significantly alter weight loss ( S2 Fig ). Taken together, this data indicates that hyperferremia, as observed in infected Tfrc Y20H/Y20H mice, does not increase peak parasitaemia. Accordingly, these findings further indicate that iron uptake by non-erythropoietic cells is decisive in the host response to malaria.

In addition to anaemia, it has been suggested that that variations in host iron levels could affect blood-stage Plasmodium parasite growth [ 51 , 52 ]. Consequently, non-haem liver iron and serum iron was measured in wild-type and Tfrc Y20H/Y20H mice upon P. chabaudi infection. At the peak of infection, both genotypes had elevated liver and serum iron levels compared to homeostasis (Figs 1J and 1K and S1). Infected wild-type and Tfrc Y20H/Y20H mice had equivalent liver iron levels ( Fig 1J ), but serum iron levels were higher in Tfrc Y20H/Y20H mice ( Fig 1K ).

While anaemia and RBC counts were comparable between both genotypes during infection, it was nevertheless possible that differences in RBC physiology could alter the course of infection. Consequently, we performed an in vitro invasion assay to determine whether Tfrc Y20H/Y20H RBCs were more susceptible to P. chabaudi invasion. Fluorescently labelled wild-type or Tfrc Y20H/Y20H RBCs were incubated in vitro with RBCs from a P. chabaudi infected wild-type mouse. Upon completion of one asexual replication cycle, invasion was assessed, and the susceptibility index was calculated ( Fig 1H ). The RBC susceptibility indices of both genotypes were comparable ( Fig 1I ), thus indicating that the higher parasite burden in Tfrc Y20H/Y20H mice was not due to a higher susceptibility of their RBCs to P. chabaudi invasion.

Anaemia-associated alterations of RBC physiology can affect malaria infection and have been put forward as the main cause of both the protective effect of iron deficiency and the increased risk associated with iron supplementation [ 10 ]. We therefore monitored RBCs in wild-type and Tfrc Y20H/Y20H mice infected with P. chabaudi. Both genotypes displayed similar levels of malaria-induced RBC loss and RBC recovery ( Fig 1E ). Moreover, Tfrc Y20H/Y20H and wild-type mice were equally severely anaemic at the nadir of RBC loss, eight days post infection (dpi) ( Fig 1F ). At the chronic stage of infection (20 dpi), however, wild-type mice showed improved recovery from anaemia compared to Tfrc Y20H/Y20H mice ( Fig 1G ), consistent with a decreased ability of the Tfrc Y20H/Y20H cells to incorporate iron.

Strikingly, mice with decreased cellular iron uptake had significantly higher peak parasitaemia and higher peak infected red blood cell (iRBC) counts ( Fig 1B and 1C ). The higher pathogen burden coincided with more severe weight loss than wild-type mice ( Fig 1D ). This phenotype contrasts previous studies, in which nutritional iron deficiency resulted in lower parasitaemia and increased survival of malaria infected mice [ 49 , 50 ]. Hence, our findings highlight a distinct role for cellular iron in malaria pathology, which acts inversely to the protective effect of anaemia. This prompted us to investigate the cause of the higher parasite burden observed in our model.

A) C57BL/6 (WT) and Tfrc Y20H/Y20H (TfR) mice were infected by intravenous (i.v.) injection of 10 5 recently mosquito-transmitted P. chabaudi infected red blood cells (iRBC). B-E) Parasitaemia (B), iRBC count (C), body weight change (D) and RBC count (E) measured throughout the course of infection. Mean ± SEM, mixed-effects analysis (B, C, E) or repeated measures two-way ANOVA (D), with Sidak’s multiple comparisons test, n = 7–9. F-G) Haemoglobin measured 8 (F) and 20 (G) days after infection. Welch’s t-test, n = 6–9. H-I) A mix of unlabelled WT RBC and iRBC were incubated with fluorescently labelled WT or TfR RBC and the invasion susceptibility index (SI) was determined after completion of a new invasion cycle. Mean, Welch’s t-test, n = 3. J-K) Liver iron and serum iron levels measured 8 days after infection. Mean, Welch’s t-test, n = 9.

Tfrc Y20H/Y20H and wild-type mice were infected with a recently mosquito-transmitted rodent malaria strain, P. chabaudi chabaudi AS, which constitutively expresses GFP (hereafter referred to as P. chabaudi) [ 46 , 47 ] ( Fig 1A ). Recently mosquito-transmitted parasites were used to mimic a natural infection more closely, as vector transmission is known to regulate Plasmodium virulence and alter the host’s immune response (48–50). Consequently, parasitaemia is expected to be significantly lower upon infection with recently mosquito-transmitted parasites, compared to infection with serially blood-passaged parasites that are more virulent [ 47 , 48 ].

To investigate the effects of cellular iron availability on the host’s response to malaria, we utilised a transgenic mouse with a mutation in the cellular iron transporter TfR1. The Tfrc Y20H/Y20H mutation decreases receptor internalisation by approximately 50%, resulting in decreased cellular iron uptake [ 29 ]. The effects of the Tfrc Y20H/Y20H mutation in erythroid cells are minimised due to a STEAP3-mediated compensatory mechanism [ 29 ]. At homeostasis, adult Tfrc Y20H/Y20H mice are healthy, normal-sized, and not anaemic ( S1 Fig ). However, they have microcytic RBCs, compensated for by an increase in RBCs ( S1 Fig ), and mildly suppressed liver and serum iron levels ( S1 Fig ).

Discussion

Iron deficiency impacts malaria infection in humans [7–9], but beyond the effects of anaemia [10], little is known about how host iron deficiency influences malaria infection. Here we investigated how restricted cellular iron acquisition influenced P. chabaudi infection in mice. TfrcY20H/Y20H mice developed comparable malaria-induced anaemia to wild-type mice, and RBC susceptibility to parasite invasion did not differ between genotypes. This therefore allowed us to largely decouple the effects of anaemia from other effects of iron on the host response to malaria. Strikingly, TfrcY20H/Y20H mice displayed an attenuated P. chabaudi induced splenic and hepatic immune response. This immune inhibition was associated with increased parasitaemia and mitigated liver pathology. Hence, for the first time, we show a role for host cellular iron acquisition via TfR1 in modulating the immune response to malaria, with downstream effects on both pathogen control and host fitness.

On first inspection, the higher parasite burden observed in TfrcY20H/Y20H mice may appear to be a severe consequence of cellular iron deficiency. In humans, however, high parasitaemia is not sufficient to cause severe disease [65]. Moreover, the risk of severe malarial disease decreases significantly after only one or two exposures, whereas anti-parasite immunity is only acquired after numerous repeated exposures [2,66]. It follows that mitigating immunopathology may be more important than restricting parasite growth for host survival. As previously noted, the TfrcY20H/Y20H mutation has relatively mild consequences for erythropoietic parameters compared to other haematopoietic lineages [29,30]. However, in humans with normal TfR1-mediated iron uptake, iron deficiency sufficient to cause immune cell iron scarcity also normally causes anaemia [67]. In such circumstances, parasite growth would likely be limited by anaemia, with the final result that iron deficiency may be protective overall, if it also minimises aspects of immunopathology.

Previous work has demonstrated the importance of regulating tissue haem and iron levels to prevent organ damage in malaria [61,62,68,69]. For example, HO-1 plays an important role in detoxifying free haem that occurs as a result of haemolysis during malaria infection, thus preventing liver damage due to tissue iron overload, ROS and inflammation [61]. Interestingly, infected TfrcY20H/Y20H mice had higher expression of Hmox1, but levels of liver iron and ROS comparable to that of wild-type mice. Consequently, this may be indicative of increased haem processing that could have a tissue protective effect. In humans, there is a correlation between transferrin saturation and ALT levels in patients with symptomatic malaria [62,70], suggesting that iron status may be linked to malaria-induced liver pathology in humans. However, it can be difficult to interpret measures of iron status in malaria infected individuals, since those parameters can be altered by inflammation and RBC destruction. Our findings reveal additional dimensions through which host iron status impacts malaria-induced tissue damage. The mitigated liver damage that we observed in P. chabaudi infected TfrcY20H/Y20H mice can likely be explained by a combination of factors; increased expression of hepatoprotective HO-1, decreased immune mediated endothelial activation, iRBC sequestration, and hepatic vascular occlusion, as well as, inhibited hepatic inflammation.

The pro-inflammatory immune response to malaria has downstream effects on cytoadherence, as pro-inflammatory cytokines activate endothelial cells, leading to higher expression of receptors for cytoadherence [2]. As a consequence, P. chabaudi infected mice that lack adaptive immunity or IFNγ-receptor signalling, have substantially decreased sequestration of iRBCs in the liver, and no detectable liver damage (as measured by ALT) [63]. Endothelial cells can also be activated by direct interactions with iRBCs [2], and in humans, ANG-2 correlates with estimated parasite biomass [59]. However, although P. chabaudi infected TfrcY20H/Y20H mice had higher peak parasitaemia, they had fewer hepatic sequestration, rosetting, and vascular occlusion events and lower ANG-2 levels. The attenuated innate and adaptive immune response is the most probable cause of decreased endothelial activation and hepatic microvascular obstruction in TfrcY20H/Y20H mice. This, in turn, likely contributed to the clearly mitigated liver pathology, in spite of the higher parasitaemia. Upon P. chabaudi infection, we observed extensive infiltration of mononuclear leukocytes into the liver, but this response was repressed in TfrcY20H/Y20H mice. Specifically, infected TfrcY20H/Y20H mice had fewer effector-like immune cells in the liver. Hepatic immune cells can contribute to liver damage in malaria, for example, by producing pro-inflammatory cytokines or through bystander killing of hepatocytes [71]. Consequently, a weaker hepatic pro-inflammatory immune response likely limited immunopathology and ameliorated malaria-induced liver damage in mice with cellular iron deficiency.

We have previously shown that hepcidin mediated hypoferremia inhibits the immune response to influenza infection in mice [21]. In influenza, cellular iron scarcity exacerbated pulmonary tissue damage, because failed adaptive immunity led to an exacerbated inflammatory response and poor pathogen control [21]. In contrast, we observed that decreased cellular iron acquisition inhibited both the innate and adaptive immune response to malaria, ultimately mitigating malaria-induced hepatic tissue damage and inflammation. This highlights the complex effects of iron deficiency on the immune system and underscores the need to consider its effect on different infectious diseases in a pathogen-specific manner. A better understanding of how host iron status affects immunity to infection could benefit the development of improved antimicrobial therapies and increase the safety of iron deficiency therapies.

The inhibited innate immune response to P. chabaudi in TfrcY20H/Y20H mice likely contributed to both the increased pathogen burden and the decreased liver pathology. Splenic MNPs are important for controlling parasitaemia [34,35,72], but MNPs are also vital for maintaining tissue homeostasis and preventing tissue damage in malaria [43,73]. Although other innate cells, such as neutrophils, NK cells and γδT cells are an important part of the immune response to malaria, only the MNP response was distinctly impaired in TfrcY20H/Y20H mice. Notably, neutrophils are known to be sensitive to iron deficiency [16,74] and to affect both immunity and pathology in malaria [75,76]. However, in the context of recently mosquito-transmitted P. chabaudi it appears that monocytes and macrophages, rather than granulocytes, may be particularly important for parasite control and tissue homeostasis [43,72].

CD4+ T cells and B cells become cell intrinsically dysfunctional during iron scarcity, as we have demonstrated in vitro. However, such cell-intrinsic effects are likely further aggravated by interactions with other iron-depleted cells in vivo. For example, CD4+ T cells support the B cell response to malaria [37,77], and the repressed CD4+ T cell response to P. chabaudi in TfrcY20H/Y20H mice presumably further constrained the B cell response. Proliferation is an aspect of immune cell function that appears to be particularly sensitive to iron deficiency [14,20,21]. Unsurprisingly, we also see the most significant inhibitory effect on immune cell populations that expand greatly during P. chabaudi infection. In addition, proliferation is often required for lymphocyte differentiation and effector function [78], and the differentiation of Tfh and Th1 cells in malaria depends on a highly proliferative precursor CD4+ T cell subset [79]. T cells from TfrcY20H/Y20H mice also had decreased KI-67 expression, further confirming impaired proliferation as a critical mechanism of immune inhibition under conditions of cellular iron scarcity. CD4+ T cells that produce pro-inflammatory cytokine are also sensitive to iron restriction, as we have shown for IFNγ, and as has been shown previously for IL-2 and IL-17 [80,81]. Interestingly, iron overload can also alter CD4+ T cell cytokine production, and excess iron can have an inhibitory effect on IFNγ production [22,82]. These observations underline that iron imbalance at either extreme can disturb immune cell function.

Despite the higher peak parasitaemia in TfrcY20H/Y20H mice, both genotypes were able to clear P. chabaudi parasites at a comparable rate and prevent recrudescence. It follows that even a weakened humoral immune response appears to be sufficient to control P. chabaudi infection. However, our study did not investigate the effects of immune cell iron deficiency on the formation of long-term immunity, which may have been more severely affected. The impaired GC response, in particular, suggests that iron deficiency could counteract the formation of efficient immune memory to subsequent malaria infections. This is in line with human observational studies that have found a link between iron deficiency and weak antibody responses to P. falciparum [7,44,45]. In humans, anti-parasite immunity forms very slowly and only after numerous repeated exposures to malaria infection [2]. Some have suggested that this effect could be explained by impaired immune cell function in malaria [83,84], and future studies should consider whether inhibited immunity as a result of iron deficiency could contribute to this phenomenon. Moreover, the extensive geographical and epidemiological overlap of iron deficiency and malaria [1,6,13] makes this concept particularly relevant for further research.

It remains to be seen what the broader importance of cellular iron is in human malaria infection, in particular within the diverse genetic context of both humans and parasites, found in malaria endemic regions. Murine models of malaria are useful in providing hypothesis-generating results, but such findings ultimately ought to be confirmed and developed further through studies in human populations. This study revealed that decreased host cell iron acquisition inhibits the immune response to malaria and ameliorates hepatic damage, despite a higher parasite load and similar degree of anaemia, in mice. Altogether, our data highlight a previously underappreciated role for host cell iron in the trade-off between pathogen control and immunopathology, and add to our understanding of the complex interactions between iron deficiency and malaria. Hence, these findings have important implications for these two widespread and urgent global health problems.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011679

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