De novo fatty-acid synthesis protects invariant NKT cells from cell death, thereby promoting their homeostasis and pathogenic roles in airway hyperresponsiveness

  1. Jaemoon Koh
  2. Yeon Duk Woo
  3. Hyun Jung Yoo
  4. Jun-Pyo Choi
  5. Sae Hoon Kim
  6. Yoon-Seok Chang
  7. Kyeong Cheon Jung
  8. Ji Hyung Kim
  9. Yoon Kyung Jeon
  10. Hye Young Kim
  11. Doo Hyun Chung

  • Curated by eLife

    eLife logo

    eLife assessment

    The study's results offer a fundamental insight into how ACC1-mediated fatty-acid synthesis affects the survival and pathogenicity of iNKT cells in allergic asthma. The inclusion of mouse models, involving genetic adjustments and reconstitution experiments, along with the disparities found in iNKT cells between allergic asthma patients and control subjects in human studies, adds compelling evidence that substantiates these findings.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Invariant natural-killer T ( i NKT) cells play pathogenic roles in allergic asthma in murine models and possibly also humans. While many studies show that the development and functions of innate and adaptive immune cells depend on their metabolic state, the evidence for this in i NKT cells is very limited. It is also not clear whether such metabolic regulation of i NKT cells could participate in their pathogenic activities in asthma. Here, we showed that acetyl-coA-carboxylase 1 (ACC1)-mediated de novo fatty-acid synthesis is required for the survival of i NKT cells and their deleterious functions in allergic asthma. ACC1, which is a key fatty-acid synthesis enzyme, was highly expressed by lung i NKT cells from WT mice that were developing asthma. Cd4 -Cre:: Acc1 fl/fl mice failed to develop OVA-induced and HDM-induced asthma. Moreover, i NKT cell-deficient mice that were reconstituted with ACC1-deficient i NKT cells failed to develop asthma, unlike when WT i NKT cells were transferred. ACC1 deficiency in i NKT cells associated with reduced expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity that promoted i NKT-cell death. Furthermore, circulating i NKT cells from allergic-asthma patients expressed higher ACC1 and PPARG levels than the corresponding cells from non-allergic-asthma patients and healthy individuals. Thus, de novo fatty-acid synthesis prevents i NKT-cell death via an ACC1-FABP-PPARγ axis, which contributes to their homeostasis and their pathogenic roles in allergic asthma.

Article activity feed

  1. Version published to 10.7554/elife.87536.4 on eLife
  2. Version published to 10.7554/elife.87536.3 on eLife
  3. eLife

    Author Response

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

    Reviewer #1 (Public Review):

    The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

    Thank you for your input into our work. Your comments have been very helpful in enhancing our work.

    Reviewer #2 (Public Review):

    In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

    Thank you for your thorough analysis of our work.

    Reviewer #1 (Recommendations For The Authors):

    1. I suggest the authors to remove one copy of the sentence "It should be noted that CD4-CreAcc1fl/fl mice lack ACC expression in both conventional CD4+ T cells and iNKT cells." in Lines 421-423.

    We have removed the redundant sentence originally shown in LINES 421-423. Thank you for pointing this out.

    Reviewer #2 (Recommendations For The Authors):

    Overall, this is a very strong study with few concerns.

    1. Are there tissue specific differences in the iNKT cell populations? The authors examined lung iNKT cells in the Figs 1-3, and used liver NKT cells for the mechanistic studies in Fig 4-5. The studies shown in Fig S2 suggest that ACC1 deficient iNKT cells have developmental defects and impaired homeostatic proliferative capacity. Does ACC1 impact lung and liver iNKT cells similarly and is the lack of allergic asthma in ACC1 deficient iNKT cells due to defective iNKT cell trafficking to the lungs or a failure to survive after transfer (Fig 3)?
    1. Similarly, are chemokine receptor expression patterns similar between WT and ACC1 deficient iNKTs (Fig 4)?
    1. The authors data suggest that Tregs are not playing a major role in the regulation of asthma induction in their ACC1 deficient mice, based on FoxP3 expression. Did the authors perform suppressor assays to show that the Tregs function similarly in WT and ACC1 deficient mice?

    In the revised manuscript, the authors addressed my major concerns.

    Thank you for your previous comments. They were very helpful in upgrading our scientific work here.

  4. eLife

    eLife assessment

    The study's results offer a fundamental insight into how ACC1-mediated fatty-acid synthesis affects the survival and pathogenicity of iNKT cells in allergic asthma. The inclusion of mouse models, involving genetic adjustments and reconstitution experiments, along with the disparities found in iNKT cells between allergic asthma patients and control subjects in human studies, adds compelling evidence that substantiates these findings.

  5. eLife

    Reviewer #1 (Public Review):

    The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

  6. eLife

    Reviewer #2 (Public Review):

    In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

  7. Version published to 10.7554/elife.87536.2 on eLife
  8. eLife

    Author Response

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

    Reviewer #1 (Public Review):

    The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

    Major concerns:

    1. This study heavily relied on the CD4-CreACC1fl/fl mice. While using of a-GalCer stimulation and Ja18KO mice mitigated the concern, it is still a major concern that at least some of the phenotype were due to the effect on conventional CD4 T cells. For example, the deletion of ACC1 gene seems also decreased the numbers of conventional CD4 T cells (Fig. 2D, Fig. S1D). Previously there were reports showing ACC1 gene in conventional CD4 T cells also plays a role in lung inflammation (Nakajima et al., J. Exp. Med. 218, 2021). If the authors believe the phenotype observed was mainly due to iNKT cells, rather than conventional CD4 T cells, a compare/contrast of the two studies should be discussed to explain or reconcile the results.

    As the reviewer pointed out, although we have experimentally demonstrated the critical role of ACC1 in iNKT cells in the regulation of allergic asthma, use of Cd4-CreAcc1fl/fl mice inevitably brings the role of conventional CD4+ T-cells in question.

    The study conducted by Nakajima et al, which reported that the absence of ACC1 in CD4+ T-cells resulted in reduced numbers and functional impairment of memory CD4+ T-cells, leading to less airway inflammation further suggests possibility of involvement of conventional CD4+ T-cells in regulation of allergic asthma. The direct compare/contrast of two studies seems difficult since Nakajima et al have focused on the role of ACC1 in memory CD4+ T cells while we have focused on iNKT cells.

    However, based on our experimental results, we believe that iNKT cells more contribute to the regulation of allergic asthma for the following reasons - (i) while the number of iNKT cells were significantly reduced in Cd4-CreAcc1fl/fl mice, the number of conventional CD4+ T cells were only slightly reduced, (ii) Cd4-CreAcc1fl/fl mice were dramatically decreased in their AHR in α-GalCer induced iNKT cell dependent allergic asthma model, and (iii) Jα18 KO mice that lack iNKT cells almost completely restore their AHR when adoptively transferred with WT iNKT cells but not ACC1-deficient iNKT cells. These results indicate that ACC1-mediated regulation of AHR is significantly dependent on iNKT cells, which might contribute to AHR in the study conducted by Nakajima et al. as well. From these, we believe that while ACC1 is a critical regulator of both conventional CD4+ T cells and iNKT cells in regulation of allergic asthma, iNKT cells may contribute more to regulation of allergic asthma compared to CD4+ T cells. We have summarized the above-mentioned contents in LINES: 421-441 with the reference you have mentioned:

    "It should be noted that Cd4-CreAcc1fl/fl mice lack ACC1 expression in both conventional CD4+ T cells and iNKT cells. It should be noted that Cd4-CreAcc1fl/fl mice lack ACC1 expression in both conventional CD4+ T cells and iNKT cells. While the use of iNKT cell- specific Cre system would demonstrate critical role of ACC1 in iNKT cells regarding allergic asthma, there is no iNKT cell-specific Cre system available yet. In addition, the study conducted by Nakajima et al, which reported that the absence of ACC1 in CD4+ T cells resulted in reduced numbers and functional impairment of memory CD4+ T cells, leading to less airway inflammation further suggests possibility of involvement of conventional CD4+ T cells in regulation of allergic asthma. However, based on our experimental results, we believe that iNKT cells more contribute to the regulation of allergic asthma for the following reasons - (i) while the number of iNKT cells were significantly reduced in Cd4-CreAcc1fl/fl mice, the number of conventional CD4+ T cells were only slightly reduced, (ii) Cd4-CreAcc1fl/fl mice were dramatically decreased in their AHR in α-GalCer induced allergic asthma model, and (iii) Jα18 KO mice that lack iNKT cells almost completely restore their AHR when adoptively transferred with WT iNKT cells but not ACC1-deficient iNKT cells. These results indicate that ACC1-mediated regulation of AHR is significantly dependent on iNKT cells, which might contribute to AHR in the study conducted by Nakajima et al. as well. From these, we believe that while ACC1 is a critical regulator of both conventional CD4+ T cells and iNKT cells in regulation of allergic asthma, iNKT cells may contribute more to regulation of allergic asthma compared to CD4+ T cells."

    1. The overall significance of the manuscript is related to the potential clinical suppression of ACC1 in human asthma patients. However, the authors only showed the elevated ACC1 genes in these patients, not even in vitro data demonstrating that suppression of ACC1 genes in the iNKT cells from patients could have potential therapeutic effect or suppression of the relevant cytokines.

    We would like to appreciate reviewer’s critical comment here. Due to paucity of iNKT cells in human PBMCs, it is extremely difficult to experimentally manipulate expression level of ACC1 in human iNKT cells. Alternatively, to address reviewer’s comment, we compared the cytokine expression of ACC1high iNKT cells from human allergic asthma patients to ACC1low iNKT cells from healthy individuals or non-allergic asthma patients. Our results show that iNKT cells from allergic asthma patients express higher levels of IL4 and IL13 than those from healthy individuals or non-allergic asthma patients, suggesting that the level of ACC1 is most likely involved in functionality of human iNKT cells as well. The results are newly shown in supplementary Fig. 5C with explanation in LINES 376-378 and 382-384:

    LINES 376-378: Lastly, the expression levels of IL4 and IL13 were significantly higher in iNKT cells from the allergic asthma patients compared to those from healthy controls and nonallergic asthma patients (Fig. S5C).

    LINES 382-384: Thus, iNKT cells from allergic asthma patients express higher ACC1, FASN and PPARG levels and lower levels of a glycolysis which is accompanied with higher levels of IL4 and IL13 than iNKT cells from healthy controls and nonallergic asthma patients.

    1. The authors report that a-GalCer administration can induce the AHR, however, in the cited paper (Hachem et al., Eur J. Immunol. 35, 2793, 2005), iNKT cell activation seems to have the opposite effect to inhibit AHR. Did the authors mean to cite different papers?

    We apologize for the confusion. We have replaced the inaccurate reference with the reference below in LINES 863-865:

    1. Glycolipid activation of invariant T cell receptor+ iNKT cells is sufficient to induce airway hyperreactivity independent of conventional CD4+ T cells, Proc Natl Acad Sci USA, 103 pp, 2782-2787 (2006),

    Reviewer #2 (Public Review):

    In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

    We are very grateful for your kind appreciation of our work.

    Reviewer #1 (Recommendations For The Authors):

    1. Related to major concern I, an iNKT cell-specific knockout of ACC1 in iNKT cells is highly desirable and should be used to directly address the question.

    As the reviewer suggested, iNKT cell-specific deletion of ACC1 will provide invaluable information to our study. Unfortunately, Cre-Loxp system that specifically targets iNKT cells has not be developed. Thus, we opted to use CD4-Cre system, which is the gold standard Cre system for the study of iNKT cells. In addition, to highlight the role of ACC1 in iNKT cells in relation to regulation of allergic asthma, we performed iNKT cell-dependent experiment models and conducted adoptive transfer of iNKT cells into iNKT cell-deficient mice (Jα18 KO). These have been discussed in the section of Discussion in LINES:421-441:

    "It should be noted that Cd4-CreAcc1fl/fl mice lack ACC1 expression in both conventional CD4+ T cells and iNKT cells. While the use of iNKT cell- specific Cre system would demonstrate critical role of ACC1 in iNKT cells regarding allergic asthma, there is no iNKT cell-specific Cre system available yet. In addition, the study conducted by Nakajima et al, which reported that the absence of ACC1 in CD4+ T cells resulted in reduced numbers and functional impairment of memory CD4+ T cells, leading to less airway inflammation further suggests possibility of involvement of conventional CD4+ T cells in regulation of allergic asthma. However, based on our experimental results, we believe that iNKT cells more contribute to the regulation of allergic asthma for the following reasons - (i) while the number of iNKT cells were significantly reduced in Cd4-CreAcc1fl/fl mice, the number of conventional CD4+ T cells were only slightly reduced, (ii) Cd4-CreAcc1fl/fl mice were dramatically decreased in their AHR in α-GalCer induced allergic asthma model, and (iii) Jα18 KO mice that lack iNKT cells almost completely restore their AHR when adoptively transferred with WT iNKT cells but not ACC1-deficient iNKT cells. These results indicate that ACC1-mediated regulation of AHR is significantly dependent on iNKT cells, which might contribute to AHR in the study conducted by Nakajima et al. as well. From these, we believe that while ACC1 is a critical regulator of both conventional CD4+ T cells and iNKT cells in regulation of allergic asthma, iNKT cells may contribute more to regulation of allergic asthma compared to CD4+ T cells."

    1. For Fig. 5A, RT-PCR verification of PPARg gene expression level change is needed.

    As suggested, we have verified the level of Pparg expression of ACC1-deficient iNKT cells through real time PCR and have added the results to Figure 5A.

    1. Verifying at least the cytokine secretion can be regulated by manipulating ACC1 expression in human asthma patient samples will make the paper much stronger.

    We would like to appreciate reviewer’s critical comment here. Due to paucity of iNKT cells in human PBMCs, it is extremely difficult to experimentally manipulate expression level of ACC1 in human iNKT cells. Alternatively, to address reviewer’s comment, we compared the cytokine expression of ACC1high iNKT cells from human allergic asthma patients to ACC1low iNKT cells from healthy individuals or non-allergic asthma patients. Our results show that iNKT cells from allergic asthma patients express higher levels of IL4 and IL13 than those from healthy individuals or non-allergic asthma patients, suggesting that the level of ACC1 is most likely involved in functionality of human iNKT cells as well. The results are newly shown in supplementary Fig. 5C with explanation in LINES 376-378 and 382-384:

    LINES 376-378: Lastly, the expression levels of IL4 and IL13 were significantly higher in iNKT cells from the allergic asthma patients compared to those from healthy controls and nonallergic asthma patients (Fig. S5C).

    Minor points:

    1. What are the cells being stained in Fig. S2C? Are they iNKT cells? If yes, why there is a tetramer-negative population?

    The density plot on the left panel of Fig. S2C represents magnetically enriched thymic iNKT cells. Due to their scarcity, thymic iNKT cells were enriched using CD1d tetramer via magnetic activated cell sorting (MACS)-based enrichment technique. After enrichment, we re-stained enriched cells with CD1d tetramers and gated out CD3 and CD1d tetramer double positive cells via flow cytometry to specifically identify iNKT cells. Due to the imperfect purity of magnetic cell separation technique, a small proportion of CD1d tetramer-negative population is seen in the left panel of Fig. S2C.

    A brief mention of this methodology has been added to the “Preparation and activation of murine T and iNKT cells” section under Materials and Methods in LINES 560-566:

    "Alternatively, thymic and liver mononuclear cells were labeled with APC-conjugated ɑ-GalCer/CD1d tetramers, bound to anti-APC magnetic beads, and enriched on a MACS separator (Miltenyi Biotec, Auburn, CA, USA; purity 89%). To analyze the development of thymic iNKTs cells, we re-stained enriched cells with CD1d tetramer and gated out CD3 and CD1d tetramer double positive cells via flow cytometry to identify thymic iNKT cells, which were used for further analysis."

    1. Where are the adoptive transferred iNKT cells purified/sorted from? Are they from lungs of Acc1fl/fl or CD4-cre/Acc1fl/fl mice, asthma-induced already? As there are very few iNKT cells in healthy and untreated mice. There is little described or explained in Methods and Materials.

    The adoptively transferred iNKT cells were purified and pooled from the lungs of at least 10 mice per group. Briefly, mouse lungs were finely chopped into small pieces using razor blades and enzymatically digested using type IV collagenase. iNKT cells from the lungs were sorted via FACS using CD1d tetramers. Approximately, 6.0 × 105 of iNKT cells were obtained from the lungs at least of 10 mice. A brief mention of this methodology was added to the “Adoptive transfer of iNKT cells in allergic asthma models” section in Materials and Methods in LINES 568-574: iNKT cells were obtained from the lungs of at least 10 Acc1fl/fl or Cd4-CreAcc1fl/fl mice. Mouse lungs were finely chopped into small pieces using razor blades and were enzymatically digested using type IV collagenase. iNKT cells from the lungs were sorted via FACS using CD1d tetramers. Approximately, 6.0 × 105 of iNKT cells were obtained from at least 10 mice and were adoptively transferred into individual recipient mouse via the intratracheal route.

    1. The use of 2-NBDG was not explained in multiple locations, particularly in Fig.5H. How is its fluorescence used to track iNKT cells? No description in Materials and methods.

    2-NBDG, a fluorescence tagged glucose analog is a indicator for measurement of glucose uptake in cells. The fluorescence intensity in 2-NBDG-treated cells represents the degree of glucose uptake in cells, which can be measured using flow cytometry. Thus, in the experiments where we treated 2-NBDG, we described the results as "glucose uptake". A brief explanation of this methodology was added to the main text in LINES 253-254. In addition, we have provided the detailed use of 2-NBDG in ‘Measurement of glucose uptake capacity’ under the section of Materials and methods in LINES 599-607: Measurement of glucose uptake capacity using 2-NBDG assay. After treating 2-NBDG, the fluorescence intensity of cells were measured using flow cytometry and represented the degree of glucose uptake in cells.

    1. Fig. 3A legends: it should be "Ja18 KO"?

    We would like to appreciate your comment on our mistake here. We have corrected this in the legend of figure 3A.

    1. There are two different mechanisms for explaining the less severe asthma/AHR phenotype in ACC1-KO iNKT cells. One is lower number of iNKT cells due to cell death, the other decreased cytokine secretions. It is not clear to the reviewer, what are the relationship between two mechanisms. Are they both contributing to the asthma phenotype or cooperative?

    As you mentioned, ACC1-deficient iNKT cells showed increase in intrinsic pathway of apoptosis as well as decrease in their cytokine secretion simultaneously. Thus, we believe that increase in cell death and decrease in cytokine expression of ACC1-deficient iNKT cells cooperatively contributed to the asthma phenotype. The above-mentioned point was discussed in LINES 453-458: Furthermore, the apoptotic tendency of the ACC1-deficient iNKT cells was accompanied by their functional impairment. The ACC1-deficient iNKT cells exhibited impaired viability and functionality. Treatment of glycolysis inhibitor in ACC1-deficient iNKT cells not only restored cellular survival but also their functionalities. From these results, we speculate that ACC1-mediated regulation of both cellular homeostasis and cytokine production cooperatively contributed to the asthma phenotype.

    Reviewer #2 (Recommendations For The Authors):

    Overall, this is a very strong study with few concerns.

    1. Are there tissue specific differences in the iNKT cell populations? The authors examined lung iNKT cells in the Figs 1-3, and used liver NKT cells for the mechanistic studies in Fig 4-5. The studies shown in Fig S2 suggest that ACC1 deficient iNKT cells have developmental defects and impaired homeostatic proliferative capacity. Does ACC1 impact lung and liver iNKT cells similarly and is the lack of allergic asthma in ACC1 deficient iNKT cells due to defective iNKT cell trafficking to the lungs or a failure to survive after transfer (Fig 3)?

    In absence of ACC1, the number of iNKT cells from both lungs and livers decreased and showed consistent features (i.e: metabolic parameters), suggesting that there was no tissue specific role of ACC1 in INKT cells.

    In the adoptive transfer experiments, we transferred equal number of WT and ACC1-deficient iNKT cells directly into mouse lungs via intratracheal route. Thus, decreased numbers of adoptively transferred ACC1-deficient iNKT cells is more likely from their intrinsically impaired homeostatic proliferative capacity, not due to defective trafficking to the lungs.

    1. Similarly, are chemokine receptor expression patterns similar between WT and ACC1 deficient iNKTs (Fig 4)?

    We compared chemokine receptor expression of WT and ACC1-deficient iNKT cells using our RNA-seq and verified their expression levels via real time q-PCR. The expression levels of these chemokine receptors were comparable between the two groups of iNKT cells. The results are newly shown in supplementary Fig. 4I with explanation in LINES 351-357:

    Meanwhile, chemokine receptor signaling is also implicated in regulating homeostasis of iNKT cell in the periphery. In particular, Meyer et al. suggested that iNKT cells require CCR4 to localize to the airways and to induce AHR. Thus, we examined the expression of several chemokine receptors, including CCR4. We found that WT and ACC1-deficient iNKT cells did not differ in their chemokine receptor expressions, suggesting that the chemokine signaling may not be critical for ACC1-mediated regulation in AHR.

    1. The authors data suggest that Tregs are not playing a major role in the regulation of asthma induction in their ACC1 deficient mice, based on FoxP3 expression. Did the authors perform suppressor assays to show that the Tregs function similarly in WT and ACC1 deficient mice?

    We would like to appreciate reviewer’s reasonable comment. However, we did not experimentally compare the suppressive capacity of WT and ACC1-deficient Tregs under the asthmatic conditions, due to minimal differences in their Foxp3 expression (Foxp3 expression is a critical determinant of suppressive function of Tregs- (Immunity. 2019 Feb 19;50(2):302-316.; Nat Immunol 2003; 4: 330–336; Cell Mol Immunol. 2015 Sep;12(5):558-65.)). Thus, we speculate that the suppressive capacity between WT and ACC1-deficient Tregs might be similar. Nevertheless, since the suppressive capacity of Tregs can also be regulated by other soluble factors and surface molecules, we cannot completely rule out the possibility that ACC1-deficient Tregs might differ in their suppressive capacity to WT Tregs in asthma. In short, while there are clear limitations to our interpretation here, we believe it is unlikely that Tregs from WT and ACC1 deficient mice show difference in their suppressive capacity during asthma. We have included above-mentioned points in the section of Discussion in LINES 415-419: In this regard, Tregs may also play a major role in asthma. However, the expression level of Foxp3 was comparable between WT and ACC1-deficient Tregs. The level of Foxp3 to some extent, serves as a critical determinant of suppressive function of Tregs. Thus, we speculate that they might not critically contribute to the development of asthma, although we cannot completely rule out the contribution of Tregs to our studies.

  9. eLife

    eLife assessment

    The study highlights an important role of key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1) in development and homeostasis of invariant natural killer T (iNKT cells), as well as its significance in asthma etiology. The work defines novel mechanisms driving metabolic regulation of iNKT cells and its role in allergic asthma. The data reported in the manuscript are convincing, and the work adds to our understanding of the metabolic regulation of iNKT cells.

  10. eLife

    Reviewer #1 (Public Review):

    The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

  11. eLife

    Reviewer #2 (Public Review):

    In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

  12. Version published to 10.1101/2023.02.15.528598v3 on bioRxiv
  13. Version published to 10.1101/2023.02.15.528598v2 on bioRxiv
  14. Version published to 10.7554/elife.87536.1 on eLife
  15. eLife

    eLife assessment

    This study by Koh et al reports an important role of key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1) in development and homeostasis of invariant natural killer T iNKT cells, as well as its significance in asthma etiology. The findings reveal that ACC1 induces de novo fatty acid synthesis via fatty acid- binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ axis in iNKT cells, which is critical for iNKT cells survival and their pathogenic roles in allergic asthma. The data reported in the manuscript are convincing, and the work adds to our understanding of the metabolic regulation of iNKT cells.

  16. eLife

    Reviewer #1 (Public Review):

    The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

    Major concerns:

    1. This study heavily relied on the CD4-CreACC1fl/fl mice. While using of a-GalCer stimulation and Ja18KO mice mitigated the concern, it is still a major concern that at least some of the phenotype were due to the effect on conventional CD4 T cells. For example, the deletion of ACC1 gene seems also decreased the numbers of conventional CD4 T cells (Fig. 2D, Fig. S1D). Previously there were reports showing ACC1 gene in conventional CD4 T cells also plays a role in lung inflammation (Nakajima et al., J. Exp. Med. 218, 2021). If the authors believe the phenotype observed was mainly due to iNKT cells, rather than conventional CD4 T cells, a compare/contrast of the two studies should be discussed to explain or reconcile the results.

    2. The overall significance of the manuscript is related to the potential clinical suppression of ACC1 in human asthma patients. However, the authors only showed the elevated ACC1 genes in these patients, not even in vitro data demonstrating that suppression of ACC1 genes in the iNKT cells from patients could have potential therapeutic effect or suppression of the relevant cytokines.

    3. The authors report that a-GalCer administration can induce the AHR, however, in the cited paper (Hachem et al., Eur J. Immunol. 35, 2793, 2005), iNKT cell activation seems to have the opposite effect to inhibit AHR. Did the authors mean to cite different papers?

  17. eLife

    Reviewer #2 (Public Review):

    In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

  18. Version published to 10.1101/2023.02.15.528598v1 on bioRxiv