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RhoH/TTF Negatively Regulates Leukotriene Production in Neutrophils
Open accessAbstract
Leukotriene B4 (LTB4) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB4 production of neutrophils within minutes, a process called “priming”. In this study, we demonstrate that GM-CSF also limits the production of LTB4 by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh−/−) mice demonstrated increased LTB4 production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB4 upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.
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TL;DR
GM-CSF induces RhoH/TTF in neutrophils, and this inducible protein acts as a negative feedback regulator that limits LTB4 production. RhoH/TTF-deficient mouse neutrophils produce more LTB4 under prolonged GM-CSF pretreatment, while cystic fibrosis patient neutrophils express more RhoH/TTF and generate less LTB4.
Key findings
- GM-CSF rapidly induces RhoH/TTF in mature neutrophils: microarray screening of 12,599 genes found RhoH/TTF upregulated 23.43-fold after 7 h GM-CSF; RT-PCR showed no detectable RhoH/TTF mRNA in freshly isolated neutrophils, induction within 30 min, and maximal mRNA at 1 h. RhoH/TTF protein was detected at 21.3 kDa, elevated between 1 and 3 h, and maximal after 4–8 h; IL-8 and IFN-γ did not induce it.
- RhoH/TTF is expressed under inflammatory conditions: blood neutrophils from cystic fibrosis patients had increased RhoH/TTF protein, and neutrophils in appendicitis and ulcerative colitis tissue expressed RhoH/TTF in cytosol and at the cell membrane.
- LTB4 production was reduced in cystic fibrosis neutrophils: normal neutrophils primed with GM-CSF and stimulated with C5a produced mean LTB4 levels >10,000 pg/ml (n = 10), whereas cystic fibrosis neutrophils produced <400 pg/ml (n = 6; p < 0.001).
- RhoH/TTF deficiency increased LTB4 under prolonged pretreatment: wild-type and Rhoh−/− mouse neutrophils produced similar LTB4 after GM-CSF priming and C5a stimulation (mean ~15,000 pg/ml), but after GM-CSF pretreatment and IL-3 priming followed by C5a, Rhoh−/− neutrophils generated significantly more LTB4 than wild-type at 3, 5, and 8 h.
Methods
- Human neutrophils were isolated from healthy donors and cystic fibrosis patients; mouse neutrophils were isolated from wild-type and Rhoh−/− C57BL/6J bone marrow. Purity was >95% for human and >90% for mouse neutrophils.
- The study used microarray gene expression profiling (HG-U95Av2), RT-PCR, quantitative real-time PCR, immunoblotting, confocal microscopy, and LTB4 ELISA. Signaling inhibitors and cycloheximide were used to test pathways and protein synthesis dependence.
- LTB4 production was measured after priming with GM-CSF or IL-3 for 30 min and subsequent C5a stimulation for 30 min; mouse experiments also used GM-CSF pretreatment for 3, 5, and 8 h. Statistical analysis used ANOVA followed by Tukey’s HSD test, with p < 0.05 considered significant.
Limitations
- The paper states that how RhoH/TTF exerts its regulatory role in neutrophils remains to be investigated, and that the molecular mechanisms of RhoH/TTF gene transcriptional activation, intracellular movement, binding partners, exact signaling mechanisms, and possible additional cellular functions remain to be investigated.
- The text makes evident that mechanistic studies in human neutrophils are limited because plasmid transfection appeared impossible and neutrophils are fragile and undergo rapid spontaneous apoptosis; the functional evidence therefore relies heavily on RhoH/TTF-deficient mouse neutrophils and cycloheximide treatment.
- Some supporting data are described as “data not shown,” and the cystic fibrosis LTB4 experiments used small groups (n = 6 for LTB4; n = 3 for immunoblot), which limits generalizability.
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49 Own result · 22 Not checked yet · 12 Own method · 12 Own interpretation
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In this study, we show that neutrophils are RhoH/TTF positive under inflammatory conditions and that RhoH/TTF negatively regulates LTB 4 production in these cells.”
- Also stated in: § Neutrophils express RhoH/TTF under inflammatory conditions · hedged
“Neutrophils expressed RhoH/TTF in these tissues, and it appeared that RhoH/TTF was located both in the cytosol and at the cell membrane (Fig. 2B).”
§ RhoH/TTF Negatively Regulates Leukotriene Production in Neutrophils 1 > N
plainstates an association- Asserted: passes — The authors put it forward as true.
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- Also stated in: § Neutrophils express RhoH/TTF under inflammatory conditions · hedged
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process.”
- Also stated in: § GM-CSF rapidly induces RhoH/TTF expression in mature neutrophils · plain
“Freshly isolated neutrophils had no detectable RhoH/TTF mRNA expression but induced RhoH/TTF mRNA upon GM-CSF stimulation within 30 min.”
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- Also stated in: § GM-CSF rapidly induces RhoH/TTF expression in mature neutrophils · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB 4 upon activation compared with normal human neutrophils.”
- Also stated in: § Neutrophils express RhoH/TTF under inflammatory conditions · plain
“Interestingly, neutrophils from patients suffering from cystic fibrosis were defective in LTB 4 production upon GM-CSF priming and subsequent C5a stimulation (Fig. 3, right panel).”
plainstates an association- Asserted: passes — The authors put it forward as true.
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- Also stated in: § Neutrophils express RhoH/TTF under inflammatory conditions · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB 4 upon activation compared with normal human neutrophils.”
- Also stated in: § Neutrophils express RhoH/TTF under inflammatory conditions · plain
“Indeed, compared with normal neutrophils, blood neutrophils from patients with cystic fibrosis had increased RhoH/TTF protein levels (Fig. 2A).”
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- Also stated in: § Neutrophils express RhoH/TTF under inflammatory conditions · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Neutrophils derived from RhoH/TTF-deficient (Rhoh ؊/؊ ) mice demonstrated increased LTB 4 production upon activation compared with normal mouse neutrophils.”
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Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the citing paper
“Rhoh -/-mice have a mild myeloid phenotype (39,40); no overt myeloid phenotype was observed in our patients.”
From Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections (2012) · cites this paper as [39]
In the citing paper
“In neutrophils, RhoH has been reported to limit cytokine-induced LTB4 production. 29”
From RhoH is a negative regulator of eosinophilopoiesis (2016) · cites this paper as [29]
In the citing paper
“RHOH expression is hardly detectable in freshly isolated human and mouse neutrophils; however, its expression is up-regulated by GM-CSF stimulation in less than 3 h (S1A Fig), confirming previously published work [27,28,38].”
From Nascent RHOH acts as a molecular brake on actomyosin-mediated effector functions of inflammatory neutrophils (2022) · cites this paper as [27]
In the citing paper
“Previously, neutrophils isolated from CF patients showed increased protein expression of RHOH compared to neutrophils from healthy donors [27].”
From Nascent RHOH acts as a molecular brake on actomyosin-mediated effector functions of inflammatory neutrophils (2022) · cites this paper as [27]
In the citing paper
“Analogous to what has been shown during lymphocyte activation, the repression of RHOH during myeloid differentiation along the monocytic pathway would be expected to release signalling through the Rho family and positively affect processes such as adhesion, chemotaxis, and trans‐endothelial migration critical to the inflammatory response [2, 4, 6, 8‐10, 12].”
From Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy (2021) · cites this paper as [9]
Citations
Cites
Neutrophils and immunity: challenges and opportunities
Nathan, C. (2006) · Nature reviews. Immunology · doi:10.1038/nri1785 · Cited by 2,971
Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis
Yousefi, S., Perozzo, R., Schmid, I., Ziemiecki, A., Schaffner, T., Scapozza, L., Brunner, T., & Simon, H.-U. (2006) · Nature Cell Biology · doi:10.1038/ncb1482 · Cited by 1,158
Self-citationRho family proteins: coordinating cell responses
Ridley, A. J. (2001) · Trends in Cell Biology · doi:10.1016/s0962-8924(01)02153-5 · Cited by 784
Ras proteins: different signals from different locations
Hancock, J. F. (2003) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm1105 · Cited by 756
Rho-family GTPases: it's not only Rac and Rho (and I like it)
Wennerberg, K., & Der, C. J. (2004) · Journal of Cell Science · doi:10.1242/jcs.01118 · Cited by 649
Roberts, A. W., Kim, C., Zhen, L., Lowe, J. B., Kapur, R., Petryniak, B., Spaetti, A., Pollock, J. D., Borneo, J. B., Bradford, G. B., et al. (1999) · Immunity · doi:10.1016/s1074-7613(00)80019-9 · Cited by 556
Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation
Ambruso, D. R., Knall, C., Abell, A. N., Panepinto, J., Kurkchubasche, A., Thurman, G., Gonzalez-Aller, C., Hiester, A., deBoer, M., Harbeck, R. J., et al. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.080074897 · Cited by 407
Martinelli, S., Urosevic, M., Daryadel, A., Oberholzer, P. A., Baumann, C. K., Fey, M. F., Dummer, R., Simon, H., & Yousefi, S. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m405883200 · Cited by 300
Self-citationConus, S., Perozzo, R., Reinheckel, T., Peters, C., Scapozza, L., Yousefi, S., & Simon, H. (2008) · The Journal of Experimental Medicine · doi:10.1084/jem.20072152 · Cited by 277
Self-citationvon Gunten, S., Yousefi, S., Seitz, M., Jakob, S. M., Schaffner, T. J., Seger, R. A., Takala, J. A., Villiger, P. M., & Simon, H. (2005) · Blood · doi:10.1182/blood-2004-10-4112 · Cited by 223
Self-citation
Cited by
Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections
Crequer, A., Troeger, A., Patin, E., Ma, C. S., Picard, C., Pedergnana, V., Fieschi, C., Lim, A., Abhyankar, A., Gineau, L., et al. (2012) · Journal of Clinical Investigation · doi:10.1172/jci62949 · Cited by 159
Dissecting Interferon-Induced Transcriptional Programs in Human Peripheral Blood Cells
Waddell, S. J., Popper, S. J., Rubins, K., Griffiths, M. J., Brown, P. O., Levin, M., & Relman, D. A. (2010) · PLoS ONE · doi:10.1371/journal.pone.0009753 · Cited by 148
Established and emerging roles for mitochondria in neutrophils
Peng, S., Gao, J., Stojkov, D., Yousefi, S., & Simon, H. (2022) · Immunological Reviews · doi:10.1111/imr.13158 · Cited by 37
Self-citationLecot, P., Ardin, M., Dussurgey, S., Alcazer, V., Moudombi, L., Pereira-Abrantes, M., Hubert, M., Swalduz, A., Hernández‐Vargas, H., Viari, A., et al. (2022) · International Journal of Cancer · doi:10.1002/ijc.33991 · Cited by 32
Gündogdu, M. S., Liu, H., Metzdorf, D., Hildebrand, D., Aigner, M., Aktories, K., Heeg, K., & Kubatzky, K. F. (2010) · Molecular Cancer · doi:10.1186/1476-4598-9-225 · Cited by 29
Metadata
- Authors
- Daryadel, A., Yousefi, S., Troi, D., Schmid, I., Schmidt-Mende, J., Mordasini, C., Dahinden, C. A., Ziemiecki, A., & Simon, H.-U.
- Affiliations
- Institute of Pharmacology
- Tiefenau Hospital Bern , Bern
- Institute of Immunology
- University of Bern (CH)
- Corresponding author
- Daryadel, Arezoo
- Troi, David
- ORCID
- Simon, Hans-Uwe
- Venue
- The Journal of Immunology
- Publisher
- Oxford University Press (OUP)
- Published
- 2009-05
- Volume
- 182
- Issue
- 10
- Pages
- 6527-6532
- ISSN
- 0022-1767 (ISSN-L)
- 1550-6606
- OpenAlex
- W1895469914
- Cited by
- 20
- References
- 43
- Citations by year
- 2024: 3 · 2023: 1 · 2022: 3 · 2021: 1 · 2020: 2 · 2016: 1 · 2015: 1 · 2012: 2
- FWCI
- 1.10
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- The Journal of Immunology (publishedVersion)
- PubMed (publishedVersion)
- BORIS (University Library Bern) (submittedVersion)
- Funders
- National Science Foundation
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (107526, 112078)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
- Health Sciences › Medicine › Pharmacology (medical) › Inflammatory mediators and NSAID effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Keywords
- leukotriene B4, neutrophils, GM-CSF, leukotriene biosynthesis, transcriptional regulation, cystic fibrosis, innate immune regulation, product inhibition
- MeSH terms
- Animals, Cystic Fibrosis, Enzyme-Linked Immunosorbent Assay, Gene Expression Regulation, Humans, Leukotriene B4, Neutrophils, Transcription Factors, Blotting, Western, Granulocyte-Macrophage Colony-Stimulating Factor, Mice, Knockout, Microscopy, Confocal, Reverse Transcriptase Polymerase Chain Reaction, Oligonucleotide Array Sequence Analysis, rho GTP-Binding Proteins, Mice
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