Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF-1α axis plays a key role in host resistance to Plasmodium infection

  1. Kely C Matteucci
  2. Nathalia PS Leite
  3. Patricia A Assis
  4. Isabella C Hirako
  5. Francielle Pioto
  6. Ogooluwa Ojelabi
  7. Juliana E Toller-Kawahisa
  8. Leonardo G Vaz
  9. Diego I Costa
  10. João S Da Silva
  11. José C Alves-Filho
  12. Ricardo T Gazzinelli

  • Curated by eLife

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    eLife Assessment

    This important study examines the role of TNF in modulating energy metabolism during parasite infection. The authors perform an elegant set of studies combining genetics, small molecule perturbation, and phenotypic experiments to highlight a role for glycolysis and glucose transport in control of parasitemia. This solid work integrates an interesting set of observations that will be of interest to the Plasmodium and pathogenesis communities with an expanded set of experiments.

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Abstract

TNF has a dual effect in Plasmodium infection, bolstering the host’s immune defense while also inducing sickness behavior. Here, we confirm that TNF signaling hampers physical activity, food intake, and energy expenditure while enhancing glucose uptake by the liver and spleen as well as controlling parasitemia in P. chabaudi (Pc)-infected mice. We also report that TNF is required for expression of inducible nitric oxide synthase (iNOS), stabilization of HIF-1α, expression of glucose transporter GLUT1 and enhanced glycolysis in monocytic cells from Pc-infected mice. Importantly, Pc-infected iNOS-/-, TNFRΔLyz2 and HIF-1αΔLyz2 mice show impaired release of TNF and glycolysis in monocytes, along with increased parasitemia and disease tolerance. Altogether, our results indicate that TNF-iNOS-HIF-1α-induced glycolysis in monocytes plays a critical role in host defense and sickness behavior in Pc-infected mice.

Tease

The role of host energy metabolism and glycolysis in monocytes as determinant of host resistance to Plasmodium infection and tolerance to disease.

Article activity feed

  1. Version published to 10.7554/elife.97759.3 on eLife
  2. Version published to 10.7554/elife.97759 on eLife
  3. eLife

    eLife Assessment

    This important study examines the role of TNF in modulating energy metabolism during parasite infection. The authors perform an elegant set of studies combining genetics, small molecule perturbation, and phenotypic experiments to highlight a role for glycolysis and glucose transport in control of parasitemia. This solid work integrates an interesting set of observations that will be of interest to the Plasmodium and pathogenesis communities with an expanded set of experiments.

  4. eLife

    Reviewer #2 (Public review):

    [Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

    Summary:

    The premise of the manuscript by Matteucci et al. is interesting and elaborates a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, that HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

    Strengths:

    The authors provide elegant in vivo experiments to characterize metabolic consequences of Plasmodium infection, and isolate cell populations whose metabolic state is regulated downstream of TNFa. Furthermore, the authors tie together several interesting observations to propose an interesting model.

    Weaknesses:

    The authors show that TNFa induces GLUT1 in monocytes, but do not show a direct role for GLUT1 or glucose uptake in monocytes in host resistance to infection.

  5. eLife

    Author response:

    The following is the authors’ response to the previous reviews

    We thank the reviewers for their careful evaluation and constructive comments throughout the two rounds of revision. We hope that the revisions have satisfactorily addressed all concerns and that the manuscript is now suitable for publication.

    This novel contribution highlights the role of this pro-inflammatory factor in the pathogenesis of and resistance to Plasmodium chabaudi infection in mice. While aspects of this response have been previously described, this study is the first to link the TNF–iNOS–HIF-1α axis to the in vivo mediation of malaria disease through its involvement in glucose metabolism. Despite well-documented metabolic alterations during malaria, including hypoglycemia and hyperlactatemia, the mechanisms underlying these changes and their relationship to host immune responses remain poorly understood. Addressing this gap is essential for elucidating how metabolic adaptation shapes disease outcomes during Plasmodium infection.

    In response to the reviewer’s comments, we have revised the Abstract, Introduction, and Discussion to clearly distinguish between:

    Previously established mechanisms (TNF–iNOS–HIF-1α–glycolysis axis), and

    The novel contribution of our study (its in vivo integration during Plasmodium infection and association with host resistance).

    Public Reviews:

    Reviewer #2 (Public review):

    Summary:

    The premise of the manuscript by Matteucci et al. is interesting and elaborates a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, that HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

    Strengths:

    The authors provide elegant in vivo experiments to characterize metabolic consequences of Plasmodium infection, and isolate cell populations whose metabolic state is regulated downstream of TNFa. Furthermore, the authors tie together several interesting observations to propose an interesting model regarding

    Weaknesses:

    The main conclusion of this work - that "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF1a axis plays a key role in host resistance to Plasmodium infection" is unsubstantiated. The authors show that TNFa induces GLUT1 in monocytes, but never show a direct role for GLUT1 or glucose uptake in monocytes in host resistance to infection (nor the hypoglycemia phenotype they describe).

    We thank the reviewer for this important comment and for highlighting the need to clarify the mechanistic link between TNF-driven metabolic rewiring and host resistance to Plasmodium infection. As noted in our first revision, our primary objective was to investigate how TNF integrates systemic and cellular metabolic responses during infection in vivo. We demonstrate that glucose uptake is significantly increased in spleen and liver during infection in a partially TNF-dependent manner, and that TNF promotes GLUT1 expression (main glucose transporter in immune cells) and glycolysis specifically in monocytic cells. Importantly, to directly address the role of TNF signaling in myeloid cells, we also observed the same phenotype (higher parasitemia, but absence of hypothermia and hypoglycemia) in mice with conditional deletion of TNF receptor 1 in lysozyme M–expressing cells (TNFR1^ΔLyz2) (Figure 4P–R), thereby validating in a cell-specific context the findings previously observed in mice with global TNFR1 deficiency. Together, these findings support a functional link between TNF signaling in monocytes, induction of GLUT1-dependent glucose metabolism, and the regulation of both systemic metabolic responses and host resistance during experimental malaria.

    While we agree that we do not demonstrate a cell-intrinsic role for GLUT1 in monocytes, multiple lines of evidence in our study support the functional relevance of glycolytic metabolism downstream of the TNF–iNOS–HIF-1α axis.

    (1) First, we show that Pc infection results in a marked increase in glucose uptake in the spleen and liver, but not in skeletal muscle or adipose tissues (Figure 2K), and that this effect is absent in TNFR-/- mice (Figure 2L), indicating a TNF-dependent and tissue-specific metabolic reprogramming. We have also clarified in the Discussion that this process appears to be insulin-independent and likely driven by pro-inflammatory signals.

    (2) Second, we show that the TNF–iNOS–HIF-1α axis. induces GLUT1 expression in monocytic cells (Figures 4M, 5D, 6L). This supports a model in which these cells contribute to observed systemic metabolic changes.

    (3) Third, we also observed a similar phenotype—characterized by higher parasitemia but absence of hypothermia and hypoglycaemia-in mice with conditional deletion of TNF receptor 1 in lysozyme M–expressing cells (TNFR1^ΔLyz2) (Figure 4P–R), thereby validating in a cell-specific context the findings previously observed in mice with global TNFR1 deficiency. These findings indicate that disruption of glycolysis phenocopies key aspects of the TNF-driven metabolic and immunological response to infection.

    (4) Finally, we demonstrate that glycolytic metabolism is functionally relevant for host resistance. Pharmacological inhibition of glycolysis in vivo using 2-DG led to increased parasitemia (Figure 6O), resembling the impaired parasite control observed in HIF-1α^ΔLyz2, TNFR-/-, and iNOS-/- mice. These findings indicate that disruption of glycolysis phenocopies key aspects of the TNF–iNOS–HIF-1α axis deficiency, supporting the conclusion that this pathway is required to sustain glycolytic metabolism and effective parasite control during infection.

    About the hypoglycemia phenotype and resistance, our previous study (PMID: 29805094) demonstrates that TNF-driven inflammation regulates systemic glucose metabolism during Plasmodium chabaudi infection. We showed that infection-induced hypoglycemia correlates with TNF levels and is associated with changes in parasite development. Specifically, leukocytes primed with IFNγ display increased expression of glucose metabolism and inflammatory genes, and TNFα-induced hypoglycemia is linked to the accumulation of non-proliferative trophozoite forms, whereas parasite replication (schizogony) occurs during host feeding. These findings indicate that blood glucose availability, regulated by TNF, directly influences parasite growth dynamics and infection outcome. Although the cellular mechanisms were not addressed in that study, our current work builds on these findings by identifying the TNF-iNOS–HIF-1α axis as a driver of GLUT1-dependent glycolysis in monocytes, linking systemic metabolic changes to a cell-intrinsic mechanism that contributes to host resistance.

    We agree that directly establishing the cell-intrinsic contribution of GLUT1 would require dedicated genetic approaches (e.g., conditional deletion in monocytes), which are beyond the scope of the present study.

    Comments on revisions:

    The demonstration that the established TNF-iNOS-HIF-1α-glycolysis axis operates in vivo during P. chabaudi infection is valuable and relevant. However, it constitutes contextual validation and must be carefully described as such. This distinction, i.e., "what has already been shown vs. what is new" is not consistently reflected in the framing of the manuscript raising overstatement concerns. This is particularly evident in the abstract and other conclusive statements, where mechanistic novelty is implied, even when the underlying pathways/mechanisms are already known. To improve the manuscript, all sentences that refer to already established findings should be accurately described as such.

    For example, the abstract states: "Here, we show that TNF signaling hampers physical activity, food intake, and energy expenditure while enhancing glucose uptake by the liver and spleen as well as controlling parasitemia in P. chabaudi-infected mice." In this sentence, the effects of TNF signaling on physical activity, food intake, energy expenditure, glucose metabolism and control of parasitemia are unequivocally established and therefore do not, in themselves, constitute new findings. Feeding behavior, not cell-intrinsic metabolism, may drive glycemic differences.

    We thank the reviewer for this comment and for highlighting the importance of distinguishing systemic metabolic effects from cell-intrinsic mechanisms. We have now revised the manuscript to more consistently distinguish between previously established mechanisms and our novel findings, particularly in the Abstract and other summary statements, to avoid any potential overstatement.

    We also would like to emphasize that, in both the Introduction and Discussion, we explicitly acknowledge that key components of the TNF–iNOS–HIF-1α–glycolysis axis have been previously described. In the Introduction, we cite studies demonstrating that TNF can induce glucose uptake and metabolic reprogramming in immune cells (refs. 14–17), as well as the role of HIF-1α as a central regulator of glycolysis and inflammation in myeloid cells (refs. 21–28). Similarly, in the Discussion, we detail prior evidence that TNF induces iNOS-derived RNI (refs. 51–54), that RNI stabilizes HIF-1α (ref. 52), and that HIF-1α drives the expression of glycolytic genes including GLUT1 (refs. 55–57). We also cite studies showing that TNF contributes to parasite control and glucose metabolism in malaria (refs. 58–61).

    Importantly, while these pathways have been described in other contexts, their integration and functional relevance in vivo during Plasmodium infection, particularly in the context of host systemic metabolism and monocytic cell function, have not been previously demonstrated. Our study addresses this gap by showing that this axis operates during P. chabaudi infection and links inflammatory signaling to both cellular metabolic reprogramming and organismal metabolic changes.

    Specifically, we demonstrate that TNF signaling drives increased glucose uptake in spleen and liver in a tissue-specific manner, promotes GLUT1 expression and glycolysis in monocytic cells, and that disruption of this axis (genetically or pharmacologically via glycolysis inhibition) impairs parasite control. In addition, we provide evidence connecting these cellular processes to systemic metabolic alterations, including hypoglycemia.

    The authors propose that TNF signaling leads to GLUT1 upregulation (in inflammatory monocytes, MO-DCs, and within the liver and spleen) during Plasmodium infection, and that this results in increased glucose uptake contributing to systemic hypoglycemia. While this is an intriguing hypothesis, we urge the authors to consider an alternative explanation that, at present, is not adequately ruled out. Given that glycemia serves as a central functional readout in the manuscript, this distinction is essential to clarify.

    The observed regulation of glycemia is likely not a direct consequence of increased glucose uptake by immune cells or by tissues but may instead reflect broader differences in disease severity across genotypes. The iNOS KO, TNFR KO, and HIF-1ΔLyz2 mice likely experience a dampened inflammatory response, which would blunt infection-induced anorexia and help preserve overall metabolic homeostasis. This alternate interpretation is supported by the authors' metabolic cage data showing increased physical activity in TNFR KO mice and the elevated food intake shown in Figure 2B.

    We thank the reviewer for this important point regarding the potential contribution of feeding behavior and systemic energy balance to the observed metabolic phenotypes. In fact, this possibility has been explicitly already incorporated into the revised manuscript. Also, we have revised the Discussion to explicitly state that the hypoglycemia observed during infection likely reflects both systemic changes in energy balance and TNF-driven metabolic reprogramming in immune cells, rather than a single isolated mechanism. Specifically, we have had already added the following statement to the Discussion:

    “Although restored physical activity, food consumption and energy expenditure in knockout mice may contribute to the observed systemic metabolic parameters by altering energy balance, these effects are not mutually exclusive with the TNF-driven, cell-intrinsic metabolic mechanisms described here”.

    In addition, we note that under naive conditions, we did not observe differences between genotypes in physical activity, food intake, energy expenditure, respiratory exchange ratio, or glycemia. These findings support that baseline metabolic parameters are comparable and that the differences observed during infection arise in the context of TNF-dependent inflammatory responses. During infection, although TNFR-deficient mice display increased food intake and activity, these differences arise in the context of altered inflammatory signaling. Therefore, rather than being mutually exclusive, behavioral and metabolic changes are likely coordinated downstream of TNF signaling.

    Furthermore, our data using pharmacological inhibition of glycolysis (2-deoxy-D-glucose) demonstrate that disruption of glycolytic metabolism results in increased parasitemia and reduced lactate levels, recapitulating key aspects of the phenotype observed in TNFR-/-, iNOS-/-, and HIF-1αΔLyz2 mice. This supports a functional role for glycolytic metabolism in host response, beyond differences in feeding behavior.

    Since anorexia and energy expenditure are tightly coupled to the inflammatory milieu, it is plausible that these behavioral and systemic differences-not monocyte nor tissue GLUT1 expression per se-are the primary contributors to the observed glycemic patterns. To support their current interpretation, the authors should perform a pair-feeding experiment in which (at least) TNFR KO mice are restricted to the same food intake as infected WT controls. This would help disentangle whether differences in glycemia truly reflect immune-driven metabolic rewiring or are secondary to differences in caloric intake.

    We thank the reviewer for this suggestion. We agree that pair-feeding experiments would provide an additional layer of control to isolate the contribution of caloric intake. However, we note that:

    (1) Baseline metabolic equivalence in naive animals argues against intrinsic differences in energy balance.

    (2) The observed phenotypes occur in the context of infection-driven inflammation, where anorexia is itself a TNF-dependent host response.

    (3) Our data support a model in which behavioral changes and metabolic rewiring are integrated components of the host response rather than independent variables.

    Importantly, our data already support a role for TNF-driven metabolic rewiring beyond feeding behavior, as inhibition of glycolysis with 2-deoxy-D-glucose recapitulates the impaired parasite control observed in genetic models. In addition, as discussed in the manuscript, systemic factors such as food intake are not mutually exclusive with cell-intrinsic metabolic mechanisms.

    We therefore consider that pair-feeding experiments are beyond the scope of the present study.

    The contribution of monocyte-specific glucose metabolism to host resistance remains unresolved.

    We appreciate the authors' effort to address the mechanistic role of glycolysis in host resistance using in vivo 2-deoxyglucose (2DG) treatment. However, I would like to point out that while this experiment is informative, it does not fully resolve the specific concern raised regarding the cell-intrinsic role of TNF-induced glycolysis in monocytes. 2DG acts systemically, inhibiting glycolysis across a wide range of cell types-including hepatocytes, endothelial cells, lymphocytes, and myeloid populations. Therefore, the observed increase in parasitemia following 2DG treatment may reflect the broad importance of glycolysis for host defense, or alternatively, may result from elevated circulating glucose levels induced by 2DG (PMID: 35841892), which could enhance parasite growth by increasing nutrient availability. Therefore, this experiment does not allow for a specific conclusion about the requirement for TNF-driven metabolic reprogramming in monocytes.

    We thank the reviewer for this comment regarding the interpretation of the 2-deoxyglucose (2DG) experiments. We agree that systemic 2DG treatment does not allow cell-specific conclusions, as it broadly inhibits glycolysis across multiple cell types. Accordingly, these data are interpreted as supporting a role for glycolysis in host defense at the organismal level, rather than as direct evidence for a monocyte-intrinsic requirement of TNF-driven metabolic reprogramming.

    At the same time, our study includes cell-specific analyses that support the engagement of this pathway in myeloid populations. In particular, we observe increased GLUT1 expression in CD11b+ cells within both the liver and spleen during infection, with marked upregulation in monocyte-derived dendritic cells (MODCs). Importantly, this induction is not observed in the corresponding knockout models, supporting the idea that TNF signaling is required for this metabolic adaptation in these cells in vivo. Consistent with this, we validated that both parasitemia and systemic glucose levels in TNFR1^ΔLyz2 mice phenocopy those observed in TNFR-deficient animals, reinforcing the contribution of myeloid TNF signaling to the metabolic and disease outcomes.

    In addition, our in vitro data demonstrate increased GLUT1 expression in WT monocytes but not in cells lacking components of the TNF–iNOS–HIF-1α axis, further supporting a pathway-specific effect. Given that GLUT1 is the primary glucose transporter in immune cells, these combined in vivo and in vitro findings, together with the 2DG experiments, provide strong evidence supporting our proposed model.

    We agree that directly establishing a monocyte-intrinsic role would require targeted genetic approaches, which are beyond the scope of the present study.

  6. Version published to 10.7554/elife.97759.2 on eLife
  7. eLife

    eLife Assessment

    This important study examines the role of TNF in modulating energy metabolism during parasite infection. The authors perform an elegant set of studies, however the evidence supporting the major claims of the manuscript is incomplete, particularly in highlighting a direct role for GLUT1 in monocytes. This work integrates an interesting set of observations that will be of interest to the Plasmodium and pathogenesis communities with an expanded set of experiments.

  8. eLife

    Reviewer #2 (Public review):

    Summary:

    The premise of the manuscript by Matteucci et al. is interesting and elaborates a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, that HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

    Strengths:

    The authors provide elegant in vivo experiments to characterize metabolic consequences of Plasmodium infection, and isolate cell populations whose metabolic state is regulated downstream of TNFa. Furthermore, the authors tie together several interesting observations to propose an interesting model regarding

    Weaknesses:

    The main conclusion of this work - that "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF1a axis plays a key role in host resistance to Plasmodium infection" is unsubstantiated. The authors show that TNFa induces GLUT1 in monocytes, but never show a direct role for GLUT1 or glucose uptake in monocytes in host resistance to infection (nor the hypoglycemia phenotype they describe).

    Comments on revisions:

    The demonstration that the established TNF-iNOS-HIF-1α-glycolysis axis operates in vivo during P. chabaudi infection is valuable and relevant. However, it constitutes contextual validation and must be carefully described as such. This distinction, i.e., "what has already been shown vs. what is new" is not consistently reflected in the framing of the manuscript raising overstatement concerns. This is particularly evident in the abstract and other conclusive statements, where mechanistic novelty is implied, even when the underlying pathways/mechanisms are already known. To improve the manuscript, all sentences that refer to already established findings should be accurately described as such.

    For example, the abstract states: "Here, we show that TNF signaling hampers physical activity, food intake, and energy expenditure while enhancing glucose uptake by the liver and spleen as well as controlling parasitemia in P. chabaudi-infected mice." In this sentence, the effects of TNF signaling on physical activity, food intake, energy expenditure, glucose metabolism and control of parasitemia are unequivocally established and therefore do not, in themselves, constitute new findings. Feeding behavior, not cell-intrinsic metabolism, may drive glycemic differences

    The authors propose that TNF signaling leads to GLUT1 upregulation (in inflammatory monocytes, MO-DCs, and within the liver and spleen) during Plasmodium infection, and that this results in increased glucose uptake contributing to systemic hypoglycemia. While this is an intriguing hypothesis, we urge the authors to consider an alternative explanation that, at present, is not adequately ruled out. Given that glycemia serves as a central functional readout in the manuscript, this distinction is essential to clarify.

    The observed regulation of glycemia is likely not a direct consequence of increased glucose uptake by immune cells or by tissues but may instead reflect broader differences in disease severity across genotypes. The iNOS KO, TNFR KO, and HIF-19775ΔαLyz2 mice likely experience a dampened inflammatory response, which would blunt infection-induced anorexia and help preserve overall metabolic homeostasis. This alternate interpretation is supported by the authors' metabolic cage data showing increased physical activity in TNFR KO mice and the elevated food intake shown in Figure 2B.

    Since anorexia and energy expenditure are tightly coupled to the inflammatory milieu, it is plausible that these behavioral and systemic differences-not monocyte nor tissue GLUT1 expression per se-are the primary contributors to the observed glycemic patterns. To support their current interpretation, the authors should perform a pair-feeding experiment in which (at least) TNFR KO mice are restricted to the same food intake as infected WT controls. This would help disentangle whether differences in glycemia truly reflect immune-driven metabolic rewiring or are secondary to differences in caloric intake.

    The contribution of monocyte-specific glucose metabolism to host resistance remains unresolved.

    We appreciate the authors' effort to address the mechanistic role of glycolysis in host resistance using in vivo 2-deoxyglucose (2DG) treatment. However, I would like to point out that while this experiment is informative, it does not fully resolve the specific concern raised regarding the cell-intrinsic role of TNF-induced glycolysis in monocytes. 2DG acts systemically, inhibiting glycolysis across a wide range of cell types-including hepatocytes, endothelial cells, lymphocytes, and myeloid populations. Therefore, the observed increase in parasitemia following 2DG treatment may reflect the broad importance of glycolysis for host defense, or alternatively, may result from elevated circulating glucose levels induced by 2DG (PMID: 35841892), which could enhance parasite growth by increasing nutrient availability. Therefore, this experiment does not allow for a specific conclusion about the requirement for TNF-driven metabolic reprogramming in monocytes.

  9. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    We now performed new experiments that were included in the manuscript. Our new results show that that monocyte-derived dendritic cells primed in vivo during P. chabaudi infection, or in vitro with TNF express high levels or GLUT-1 (Figures 4M, 5D, 6L). Furthermore, our new data show that mice treated with 2-DG (na inhibitor of glycolysis) are more susceptible to infection (Figures 6N, O). In addition, new results of glucose uptake by muscle and adipose tissues were added to the manuscript. Finally, figure legends were revised, densitometric analysis performed, and other issues addressed in the text.

    Please see below a point-by-point reply to the Reviewers’ comments.

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    The manuscript by Kely C. Matteucci et al. titled "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF-1α axis plays a key role in host resistance to Plasmodium infection" describes that TNF induces HIF-1α stabilization that increases GLUT1 expression as well as glycolytic metabolism in monocytic and splenic CD11b+ cells in P. chabaudi infected mice. Also, TNF signaling plays a crucial role in host energy metabolism, controlling parasitemia, and regulating the clinical symptoms in experimental malaria.

    This paper involves an incredible amount of work, and the authors have done an exciting study addressing the TNF-iNOS-HIF-1α axis as a critical role in host immune defense during Plasmodium infection.

    Reviewer #2 (Public Review):

    Summary:

    The premise of the manuscript by Matteucci et al. is interesting and elaborates on a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

    Strengths:

    The authors provide elegant in vivo experiments to characterize metabolic consequences of Plasmodium infection, and isolate cell populations whose metabolic state is regulated downstream of TNFa. Furthermore, the authors tie together several interesting observations to propose an interesting model.

    Weaknesses:

    The main conclusion of this work - that "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF1a axis plays a key role in host resistance to Plasmodium infection" is unsubstantiated. The authors show that TNFa induces GLUT1 in monocytes, but never show a direct role for GLUT1 or glucose uptake in monocytes in host resistance to infection (nor the hypoglycemia phenotype they describe).

    We kindly disagree with the Reviewer. There is a series of experiments showing that TNFR KO (Figures 1, 2, 4), HIF1a KO (Figure 5) and iNOS KO (Figure 6) mice have partially impaired inflammatory response and control of parasitemia (Figures Figures 1E, 5G and 6B).

    To further address the issue raised by the reviewer, we performed two sets of experiments. First, we show, in vitro, the impact of TNF stimulation on GLUT1 expression and glucose uptake (Figure 4M, 5D, 6L). Our results show that GLUT1 is increased after 18 hours with TNF (100 ng/mL) stimulation in MODCs from WT mice but not from iNOS KO, HIF1a KO e TNFR KO mice. Similar results were obtained with monocytic cells derived from infected mice (Figure 4L, 5C, 6K). The results support the discussion by demonstrating that TNF stimulation influences GLUT1 expression in monocytic cells. This aligns with the proposed mechanism that TNF signaling regulates HIF-1α stabilization and glycolytic metabolism via RNI. The absence of GLUT1 upregulation and glucose uptake in TNFR KO, iNOS KO and HIF-1α KO mice further reinforces the role of RNI in promoting HIF-1α stabilization, as suggested in the discussion.

    Recommendations for the authors:

    Reviewer #1 (Recommendations For The Authors):

    Major points

    All Figure legends are not precise about the data express means {plus minus} standard errors of the means (SEM) or SD. Figure 1D shows no SD in the data from the uninfected group. It strongly suggests precise and improving all figure legends, giving more details in terms of including an explanation of all symbols, non-standard abbreviations, error bars (standard deviation or standard error), experimental and biological replicates, and the number of animals, and representative of the independent experiments.

    We apologize for the lack of details in the Figure legends. As requested, we are now indicating whether we used SEM or STDV, number of mice per group, number of replicate experiments. We also clarified the groups that are being compared, and the statistical significance indicated by the symbols. We also standardized symbols as asterisk only, and number of asterisk indicating the significance.

    Figure 1. The figure legend has no information about the organ for which TNF mRNA was measured (Figure 1D). Also, regarding the TNF data, Figure 1 C e 1D shows that the circulating levels of TNF and the expression of TNF mRNA in the liver peaked at the same time point, and after 6h, there is no difference between infected and uninfected mice. It would be expected that the TNF mRNA expression would be detected earlier than the protein, assuming that the primary source of TNF is from the liver. Is there another organ that could mainly source blood TNF levels? Did the authors have a chance to measure the blood TNF levels during infection (0-8dpi), besides the measurement at different times only on day 8?

    We included in the legend of Figure 1D that mRNA was extracted from liver.

    Liver and spleen are the main reservoir of infected erythrocytes and the main source of cytokines during the infection with the erythrocytic stage of malaria. The results presented in Figures 1C and 1D are from in vivo experiments, not a controlled cellular experiment in vitro. So, we can not conclude about exact time and synchronous production of TNF mRNA and protein. We have published earlier that during P. chabaudi infection, the peaks of TNF mRNA expression and the levels of circulating TNF protein occur between midnight and 6 am (Hirako at al., 2018). Hence the results are consistent in the results described here. In addition, this earlier study also shows that the same pattern of TNF at days 6 and 8 post-infection are similar. Furthermore, in another studies, we reported that the peak of TNF production occurs between days 6 and 10 post P. chabaudi infection (Franklin et al, PNAS, 2009; Franklin et al, Microbes and Infection, 2007). This is now clarified in the text (page 05, line 132):

    “As previously demonstrated, the circulating levels of TNF and expression of TNF mRNA in the liver peaked at 6 am (end of dark cycle) at 8 dpi (Figure 1C and 1D), and has been reported to peak between days 6 and 10 post-infection, with a consistent pattern observed on days 6 and 8.”

    Figure 2. "We observed that in naïve animals, all of these parameters were similar in TNFR-/- and C57BL/6 mice (Figures 2A-D, top panels, and Figures 2E-H)." Interestingly, the respiratory exchange rate of TNFR-/- uninfected mice seems higher in TNFR-/- uninfected mice than in naïve uninfected mice, and this pattern seems to be more pronounced in TNFR-/- uninfected mice. Is there any suggestion that could explain the change in respiratory exchange rate behavior without infection in those animals?

    At the moment, we have not investigated the basis of this difference between uninfected WT and TNFR KO mice, which goes beyond the scope of this research. This is indeed an interesting observation that should be pursued in the future by our group and elsewhere. We mentioned this difference, when describing the results (page 06, lines 155):

    “We observed that in naïve animals, all of these parameters were similar in TNFR-/- and C57BL/6 mice (Figures 2A-D, top panels and Figures 2E-H), with a slightly higher respiratory exchange rate in uninfected TNFR-/- mice. In contrast, all the evaluated parameters were decreased in infected C57BL/6 mice compared to their naïve counterparts during the light and dark cycles. When we analyzed only infected mice, the alterations in all parameters were milder in TNFR-/- compared to C57BL/6 mice (Figures 2A-D bottom panels and 2E-H).”

    Figure 3. To give an idea of the main population of non-parenchymal cells, it will be helpful to clarify briefly how non-parenchymal cells from the liver of infected or uninfected mice were isolated.

    We described in detail at Material and Methods (Page 19, Lines 566.)

    Figure 3, B, C, D, G and Figure 4K and Figure 5 A and B - Semi-quantitative data through the densitometric analysis of western blots should be included in all figures.

    Thank you for the suggestion. We now included the densitometric analysis for all Western blot results in Supplementary figure.

    Figure 4. The author describes, "We observed that except for Hexokinase-3, the expression of mRNAs of glycolytic enzymes (Hexokinase-1, PFKP, and PKM) was increased in C57BL/6 but not TNFR-/- 8dpi." Sometimes, it is hard to understand which groups have been compared to some data. Be precise in describing the statistical analysis between the groups. It seems that those genes were increased in "infected C57BL/6 in comparison to uninfected mice, but not TNFR-/- 8-dpi. Moreover, even though the authors include statistic symbols "ι, ιι, ιιι" in other legends, there is no explanation about statistic symbols in the legend of Figure 4.

    As mentioned above, we improved the descriptions of all figures in the legend, and when necessary in the main text describing the results.

    Figure 5. The authors describe, "We found that GLUT1 protein and glycolysis (ECAR) was impaired, respectively, in monocytic cells and splenic CD11b+ cells from infected, as compared to uninfected HIF-1aΔLyz2 mice (Figures 5C-5E)." The GLUT-1 expression was inhibited in both cells compared to HIF-1afl/fl mice but not even close to impaired GLUT-1 expression. There is still a robust amount of GLUT-1 expression, and significantly higher when compared to cells from uninfected mice.

    We tuned our statement to partially impaired, indicating that other host or parasite components maybe be also influencing GLUT-1 expression. In fact, we have recently published that IFNγ has also an important role in regulating GLUT1 expression in MO-DCs and this reference is mentioned in the text (page 10, line 291):

    “We found that glycolysis (ECAR) and GLUT1 expression were impaired, though partially, in monocytic and splenic CD11b+ cells from infected HIF-1aΔLyz2 mice (Figures 5C-5E) compared to infected WT mice. The level of GLUT1 expression that is still maintained is likely due to other host or parasite factors, such as IFN-γ (Ramalho 2024).”

    Figure 6. It is essential to have more information about the number of replicates in Figure 6A. However, there are just two dots replicates in the condition CD11b+ splenic cells from C57BL/6 stimulated with or without LPS (purple bars). It is essential to be precise regarding the number of experimental and biological replicates in each experiment and the statistical analysis that has been applied, including this group. Furthermore, the author concludes, "...these data demonstrated that RNI induces HIF-1α expression...." This conclusion needs a more careful description since no data supports that monocytic cells or splenic CD11b+ cells from iNOS-/- infected mice decrease stabilization of HIF-1αm using blotting, as shown in Figure 5 A.

    As mentioned above the number of replicates for each experiment was included in the figure legends.

    Minor Points.

    Figure 3. "Hepatocytes have an important role in glucose uptake from the circulation, and they do this primarily through GLUT2 (38), whose mRNA expression was downregulated (Figure 3A) and protein expression unchanged in response to Pc infection (Figure 4K)." I suggest moving the Figure 4K to Figure 3 to make it easy to follow the data description.

    We thank the reviewer for the suggestion. However, we chose to keep Figure 4K in Figure 4, as this panel includes data from TNF receptor deficient mice, and the analysis of TNF knockout models is first introduced and discussed in Figure 4. For clarity and consistency, we therefore maintained this panel within Figure 4.

    Line 433. Replace iNOS for iNOS-/- mice.

    iNOS is now replaced for iNOS-/- mice.

    Reviewer #2 (Recommendations For The Authors):

    The premise of the manuscript by Matteucci et al. is interesting and elaborates on a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

    The main goal of this work is to study the interplay of TNF/HIF1a/iNOs in the pathogenesis in an experimental model of malaria. To dissect the molecular mechanism by which TNF induces reactive nitrogen species and regulates HIFa expression is beyond the scope of our research. Nevertheless, there is a vast literature addressing these issues. We now include in the discussion a paragraph describing the main conclusion of these studies published previously (page 12, line 363):

    "Previous studies have shown that TNF induces the production of RNI through the upregulation of iNOS via the NF-κB pathway (63, 64). TNF-mediated iNOS expression is critical for NO production, which in turn stabilizes HIF-1α by inhibiting prolyl hydroxylases (PHDs) even under normoxic conditions (58, 59). HIF-1α then upregulates the expression of glycolytic genes, including GLUT1 (22, 62).”

    Major comments

    Issues concerning novelty

    Some of the reported observations are not novel. TNFa and TNFa signaling has been demonstrated to contribute to the release of certain cytokines, and to contribute to the control parasitemia (PMID: 10225939). TNFa has been shown to increase glucose uptake in tissues (PMID: 2589544). There is a textbook about the role of INOS during the pathogenesis of malaria, including its association with parasite control (https://link.springer.com/chapter/10.1007/0-306-46816-6_15). Furthermore, other mechanisms controlling glycemia during Plasmodium infection have been shown (PMID: 35841892). The authors should adequately discuss other papers which have reported some of their findings.

    Thanks for the comments on previously existing literature. We are well aware of some of this earlier literature. Some of these earlier findings are mentioned in our manuscript. We emphasized these fundamental findings in the discussion, as requested (page 12, line 368):

    “TNF has been described as a critical mediator in malaria, driving cytokine release and parasitemia control (PMID: 10225939). It also enhances glucose uptake in tissues, aligning with our findings of increased glycolysis in monocytes (PMID: 2589544). The role of iNOS in malaria is well documented. IFN-γ and TNF induced the production of NO, which inhibits parasite growth but can cause tissue damage and organ dysfunction, especially in severe malaria (Mordmüller et al., 2002). Recent studies also highlight the complexity of glycemia regulation during Plasmodium infection describing its role in modulating parasite virulence and transmission (PMID:35841892). These studies demonstrate the critical function of TNF and iNOS in immune responses against Plasmodium, aligning with our findings of this axis and metabolic rewiring that are essential for monocyte activation and outcome of Pc infection.”

    The authors claim that "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF1a axis plays a key role in host resistance to Plasmodium infection," and contributes significantly to their effector functions (particularly parasite clearing), and the systemic drop in glycemia observed during Pc infection. Although the authors show that TNFa does result in altered metabolism and increased GLUT1 levels in a subpopulation of monocytes, the evidence that TNFa-induced glylcolysis plays a key role in host resistance is correlative at best.

    This is an important question. We did show that TNFR KO have higher parasitemia. But TNF is pleiotropic cytokine and has multiple roles on innate and acquired immunity. The experiment we have performed and helps to address this issue is the in vivo treatment with 2DG. We found that treatment with this inhibitor of glycolysis results in a increase of parasitemia. These results are now included in Figure 6.

    When considering that the majority of monocytic populations are reduced in frequency and only a small subset (i.e., Monocyte-derived DCs) increase in frequency (Fig 3K) during Pc infection, this makes it very difficult to demonstrate that a cell population whose overall frequency reduces contributes significantly to the drop in glycemia during Pc infection. The authors should therefore include experiments that demonstrate that the inhibition of glycolysis induced by TNFa in monocytes is protective and/or contributes to a decrease in extracellular glucose. The authors could assess the impact of the loss of function of GLUT1 on activated monocytes and monocyte-derived DCs on glycemia upon TNFa stimulation.

    We agree. We focused on monocytes and the derived inflammatory monocytes and MO-DCs. In fact, the frequency of monocytes, considering the inflammatory monocytes and MO-DCs, is increased both in spleen and liver. One interesting result is that the HIF1a Lysm KO mice has impaired metabolism, attenuated hypoglycemia and increased parasitemia (Figure 5). Nevertheless, we agree that our current data thus not proof that the glycemia is due to the consumption of glucose by the activated monocytes, and that these are the only cells with increased glucose consumption. This is now added to the discussion (page 13, line 395):

    "Although the frequency of MO-DCs increases during infection, other cell populations may also contribute to glucose consumption. Further experiments, including the assessment of GLUT1 function in these populations, are needed to clarify their contribution to glucose consumption during infection."

    Furthermore, in the current state of the manuscript, it is unclear how activated monocyte populations uptake glucose. The authors claim that glucose uptake by activated monocytes is GLUT1-dependent, however, glucose transport via GLUT1 is insulin-dependent. Since Plasmodium infection is associated with insulin resistance, and almost unquantifiable levels of insulin (PMID: 35841892), and TNFa itself induces insulin resistance (PMCID: PMC43887), it is unclear how the activated monocyte population uptakes glucose. If the authors consider TNFa to be sufficient for GLUT1 induction, in vitro experiments (TNFa+monocytes) could bolster this claim (and support that GLUT1 is induced in an insulin-independent mechanism.

    There is significant evidences indicating that in contrast to GLUT4, induction of GLUT1 in mice is independent of insulin (PMID: 9801136). In our case, seems to be induced by the cytokines TNF and IFN𝛾(this study and Ramalho et al., 2024). We now performed experiments exposing monocytes to TNF and evaluating GLUT1 expression. The results indicate that monocytes exposed to TNF (100 ng/mL) for 18 hours from WT mice exhibited a significant increase in GLUT1 expression. This increase was comparable to the increased-GLUT1 phenotype observed in infected animals. The results of this experiment were included in the manuscript.

    A text was included to the discussion to clarify the issue of insulin dependence of GLUT1 expression (page 13, line 388):

    “GLUT1 expression is recognized as independent of insulin, in contrast to GLUT4 (PMID: 9801136). In our model, this regulation appears to be driven by pro-inflammatory cytokines, particularly TNF. Supporting this, our results show that in vitro stimulation with TNF, significantly increases GLUT1 expression in monocytes, accordingly to the ex vivo phenotype observed in infected animals.”

    Alternative hypothesis which might explain their phenotypes

    Figure 2 A-H: The metabolic effects of the genetic manipulations including INOS KO, TNFR KO, and HIF-1α∆Lyz2 could be explained by lesser disease morbidity owed to a reduction of inflammatory response during infection. Under this condition, the development of anorexia will not be as profound in the knock-outs compared with wild-type littermate controls, since anorexia of infection is tightly linked to the magnitude of inflammatory response. Accordingly, infected knock-out animals can keep eating, which presumably impacts glycemia, maintenance of core body temperature, and overall energetics of infected mice. The authors should exclude this possibility.

    We consider this possibility and the discussion now elaborates about this alternative hypothesis. We believe, that these two mechanisms are not mutually exclusive (page 16, line 474):

    “Although restored physical activity, food consumption and energy expenditure in knockout mice may contribute to the observed systemic metabolic parameters by altering energy balance, these effects are not mutually exclusive with the TNF-driven, cell-intrinsic metabolic mechanisms described here.”

    Minor comments

    The authors showed increased parasitemia upon TNFR and HIF1a depletion in the LyZ2 compartment. The same was observed upon organismal INOS depletion. This raises the question of whether the TNFHIF-INOS signaling axis is adaptive or maladaptive during Pcc infection. The authors should show host survival in mice lacking TNFR and HIF1a in the LyZ2 compartment, and in mice lacking INOS (presumably, they have these data).

    Despite the fact the various knockout mice have increased parasitemia and signs of disease, they all survive the infection. This is now included in the Figure legends.

    Are the higher tissue glucose levels specific to the liver and the spleen or this is a more general event? Have the authors looked at other organs?

    We now added the results of glucose uptake in the muscle and adipose tissues in figure 2. The fact that the glucose uptake is not increased in muscle and adipose tissue, further suggest that the increased glucose uptake in this model is insulin independent.

    Figure 1F: All core body temperatures are within the physiological range, i.e., >36 degrees C. This makes it unclear why the authors regarded this as hypothermia. The authors should present experiments demonstrating the development of hypothermia in Figure 1F, as they claim this.

    Temperature changes in mouse kept in animal house have been an issue discussed in the field. It is clear, however, that early in the morning (end of active period) mice have torpor. Lower temperature and physical activity.

    In Figure 4, since the authors already suggested that extra-hepatic cells, and not the liver parenchyma, contribute to glucose uptake, the authors should clarify why they analyzed the whole liver in Figure 4, and not extra-hepatic cells. Furthermore, the authors should quantify the hepatic monocytic population in non-infected versus infected wild-type animals.

    The reason we used whole liver, is that the number of non-parenchymal cells obtained from liver is limited for Western blot analysis. We thought that was important to show that expression of GLUT1 was decreased in the liver of TNFR KO mice. Nevertheless, the level of TNFR expression in different cell types in the liver was shown by flow cytometry. In addition, we performed the WB with cells extracted from the spleen, where lymphoid and myeloid cells are more abundant.

    Line 87: Phagocytizing parasitized what?

    This has been corrected in the manuscript.

    Line 111 Define RNI before being used.

    Is there a gender disparity in the TNFR KO phenotype? If yes, the authors should comment about this in their discussion.

    This has been defined and addressed in the manuscript

    Line 192: Did the authors mean 3B??

    In 3M, please plot monocytes from uninfected animals.

    The plot of uninfected animals are now included in Figure 3M

    Line 390 Remove the extra dash in HIF1a.

    Extra dash has been removed.

    Line 397 Define RA

    RA is now defined.

  10. Version published to 10.7554/elife.97759.1 on eLife
  11. eLife

    eLife assessment

    This important study examines the role of TNF in modulating energy metabolism during parasite infection. The authors perform an elegant set of studies, however the evidence supporting the major claims of the manuscript is incomplete. This work integrates an interesting set of observations that will be of interest to the Plasmodium and pathogenesis communities with an expanded set of experiments.

  12. eLife

    Reviewer #1 (Public Review):

    Summary:

    The manuscript by Kely C. Matteucc et al. titled "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF-1α axis plays a key role in host resistance to Plasmodium infection" describes that TNF induces HIF-1α stabilization that increases GLUT1 expression as well as glycolytic metabolism in monocytic and splenic CD11b+ cells in P. chabaudi infected mice. Also, TNF signaling plays a crucial role in host energy metabolism, controlling parasitemia, and regulating the clinical symptoms in experimental malaria.

    Weaknesses:

    Even though iNOS deficiency reduced the expression of the glycolytic enzymes as well as reduced GLUT1 expression and lower ECAR in splenic monocytes, there is no data to support that RNI induces the expression and stabilization of HIF-1α.

    This paper involves an incredible amount of work, and the authors have done an exciting study addressing the TNF-iNOS-HIF-1α axis as a critical role in host immune defense during Plasmodium infection.

  13. eLife

    Reviewer #2 (Public Review):

    Summary:

    The premise of the manuscript by Matteucci et al. is interesting and elaborates on a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

    Strengths:

    The authors provide elegant in vivo experiments to characterize metabolic consequences of Plasmodium infection, and isolate cell populations whose metabolic state is regulated downstream of TNFa. Furthermore, the authors tie together several interesting observations to propose an interesting model.

    Weaknesses:

    The main conclusion of this work - that "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF1a axis plays a key role in host resistance to Plasmodium infection" is unsubstantiated. The authors show that TNFa induces GLUT1 in monocytes, but never show a direct role for GLUT1 or glucose uptake in monocytes in host resistance to infection (nor the hypoglycemia phenotype they describe).

  14. Version published to 10.1101/2024.03.26.586751 on bioRxiv