Neutrophil KLF2 regulates inflammasome-dependent neonatal mortality from endotoxemia

Background

Babies born too early are still developing their immune system, and therefore, cannot efficiently fight off bacterial infections, neonatal sepsis continues to be a major cause of death. The immune response to bacterial infections in neonates is mediated primarily by myeloid cells. Neutrophils are the most abundant type of myeloid cells. In neonates, neutrophils have reduced migration capabilities and demonstrate impaired functionality in combating bacterial infections. Also, they produce increased levels of pro-inflammatory cytokines, which can contribute to a lethal cytokine storm [3-5], a defining feature of sepsis-induced organ failure. Endotoxemia is often used as a model for the hyperinflammation associated with sepsis. Endotoxemia occurs when there are elevated levels of endotoxins, such as lipopolysaccharides, into the bloodstream.

Krüppel-like factor-2 (KLF2), belonging to the zinc-finger family of DNA-binding transcription factors, plays a key role in the immune dysregulation observed in preterm newborns. KLF2 regulates the hosts response to polymicrobial infection and endotoxic shock [6]. However, how KLF2 is regulated during development and how it affects the activation of the NLRP3 inflammasome in preterm newborns is still unknown. In this preprint, Mukherjee and team show that KLF2 neutrophil gene expression is age-dependent and is involved with pro-inflammatory responses. Overall, they show that KLF2 is a promising target in successfully treating newborns with sepsis.

Key Findings

Neutrophil KLF2 regulates LPS-induced mortality in an age-dependent manner

Through the use of a myeloid-specific Klf2 knockout murine model of endotoxemia, the preprint authors were able to study how the decrease in myeloid Klf2 gene expression is responsible for the increased mortality in preterm neonates. They found that among the preterm neonates with the Klf2 gene knocked out there was a much lower survival rate from sepsis compared to the control pups. Knowing this, Mukherjee and colleagues then looked at older pups and found that all these recovered from sepsis, regardless of the genotype. This told them that their observation must be age dependent.

Mukherjee and team then decided to take pups with 50% neutrophil depletion (P3-Ly6G) and pups with 73% neutrophil depletion (P1-3 Ly6g) and inject them with LPS on day 4 (P4) to induce endotoxemia. Upon doing this, they found that among the P3-Ly6G pups there was a significant increase in survival in those pups with the Klf2^fl/fl/Lyz2^Cre genotype. After injecting the P1-3 Ly6G pups with LPS, there was a significant increase in survival for both genotypes. This indicates that the neutrophil KLF2 is critical in regulating the age-dependent mortality rate.

Myeloid-KLF2 regulates the age-dependent pro-inflammatory response

The preprint authors next wanted to know if the neutrophil KLF2 also regulates the pro-inflammatory response in an age-dependent manner. In order to study this, they collected sera 4 hours after the pups were exposed to either PBS (control) or LPS (endotoxic shock). When comparing the two genotypes once again, they found that there was a greater pro-inflammatory response in the Klf2^fl/flLyz2^Cre pups when compared to the Lyz2^Cre pups. The pro-inflammatory cytokine IL-1ß was the most significantly and consistently elevated cytokine in P4 Klf2^fl/flLyz2^Cre pups, and the anti-inflammatory cytokine IL-4 was decreased in these pups. The authors concluded that the myeloid Klf2 deletion is associated with the exaggerated pro-inflammatory response in neonates which is much more pronounced at the earlier neonatal ages. This shows the myeloid-KLF2 is not only involved in regulating the pro-inflammatory response, but is also an age -dependent factor.

Neutrophil-KLF2 Affects NLRP3 Inflammasome Priming

KLF2 is essential for controlling NLRP3 inflammasome and the subsequent release of IL-1ß. The authors compared control pups with pups with KLF2 knocked out pups and found there was a much greater baseline and NLRP3 protein expression in the pups without KLF2. This rise is linked to more cleavage of pro-IL-1ß and increased mature IL-1ß into the supernatant. Interestingly, there were no discernible changes in Nlrp3 mRNA levels seen despite the increase in NLRP3 protein expression, which indicates a non-transcriptional method of regulation. Additionally, the age-dependent rise in NLRP3 protein expression and IL-1ß release is reversed with overexpression of KLF2, highlighting the function of KLF2 in inhibiting the inflammasome activity.

Together, these findings show that neutrophil-KLF2 contributes to the elevated inflammatory response in neonates by acting as a developmental regulator of NLRP3 inflammasome priming and activation.

Postnatal Day 4 Mortality is Dependent on NLRP3 Inflammasome Activation

This study offers strong evidence that NLRP3 inflammasome activation plays a critical role in newborn mortality from endotoxemia.  Compared to control Lyz2^Cre pups, mortality in P4 Klf2^fl/flLyz2^Cre pups increased significantly after LPS exposure.  In Klf2^fl/flLyz2^Cre pups, inhibition of NLRP3 with MCC950 dramatically increased survival rates, lowering mortality from 80% to 7%.  The effectiveness of NLRP3 inhibition was further supported by comparable drops in IL-1β blood levels.  Furthermore, IL-1 receptor antagonism (IL-1ra) provided strong protection, resulting in almost total survival for both genotypes.  These results support the idea that the pathophysiology of newborn endotoxemia is mostly dependent on NLRP3-mediated IL-1β generation.

Crucially, in this model, neutrophils were found to be the primary cause of death.  MCC950 treatment of bone marrow neutrophils in-vivo inhibited the release of IL-1β, indicating that the in-vivo survival effect was probably due to neutrophil-specific suppression of NLRP3. NLRP3 suppression could provide a viable therapeutic method for neonatal sepsis, as these findings collectively demonstrate that NLRP3 inflammasome activation in neutrophils is a significant predictor of neonatal endotoxemia-related mortality.

What we like about this preprint

This study provides insight into how Krüppel-like factor 2 (KLF2) is involved in sepsis in preterm neonates. We liked that the authors of this study used many different methods to approach this issue from different angles. It provides very convincing information on the age dependency, the different pathways involved, and the different genotypes that may influence the survival rate. We were also drawn to this study because of how well thought out the experiments were, and how much information was provided on how to increase the survival rate of preterm neonates.

Future Directions

Future research should investigate the molecular mechanisms by which KLF2 controls neutrophil NLRP3 inflammasome priming. Further understanding of KLF2’s immunomodulatory function may be gained by examining whether it directly interacts with important transcriptional regulators or signaling pathways involved in inflammasome activation.  Also, in vivo models of inflammatory diseases like sepsis or autoimmune disorders may help uncover the physiological importance of KLF2-mediated inflammasome control and its potential as a therapeutic target.  Gaining insight into how KLF2 affects neutrophil function in different tissues may also have wider ramifications for innate immunity and inflammation resolution.

Future studies should also concentrate on determining the upstream triggers and downstream effectors that contribute to the observed lethality with respect to the involvement of NLRP3 inflammasome activation in postnatal mortality.  It may be possible to get important knowledge about newborn immunological vulnerabilities by identifying if particular inflammatory mediators or environmental variables worsen NLRP3 activation during early postnatal development.  Furthermore, pharmacological or genetic therapies that target the inflammasome pathway might offer viable methods to reduce the risk of death.  Potential prenatal implications on early-life inflammatory responses may also be elucidated by more research into the interactions between maternal immunological variables and neonatal inflammasome activation.

Questions for the authors

1. Have you explored whether pharmacological agents that modulate KLF2 or inflammasome activation can rescue the neonatal lethality observed in your models?
2. Would you test the KLF2 function in any in vivo models of neonatal sepsis or autoimmune disease to validate the translational relevance of your findings in the future?
3. Have you identified any specific transcriptional regulators or signaling pathways that KLF2 interacts with to suppress NLRP3 inflammasome priming in neonatal neutrophils?
4. Do you have any evidence that KLF2 functions through metabolic regulation or redox-sensitive pathways to exert its immunosuppressive effects in neonatal neutrophils?
5. Based on your findings, do you envision KLF2 or its regulatory pathways being feasible therapeutic targets for mitigating sepsis-related mortality in neonates?
6. Have tissue-specific knockout models been used or considered to explore whether KLF2 regulation in neutrophils varies across different organs during inflammation?

References

[1] Stoll, B. J., Puopolo, K. M., Hansen, N. I., Sanchez, P. J., Bell, E. F., Carlo, W. A., Cotten, C. M., D’Angio, C. T., Kazzi, S. N. J., Poindexter, B. B., Van Meurs, K. P., Hale, E. C., Collins, M. V., Das, A., Baker, C. J., Wyckoff, M. H., Yoder, B. A., Watterberg, K. L., Walsh, M. C., Devaskar, U., Laptook, A. R., Sokol, G. M., Schrag, S. J., Higgins, R. D., Eunice Kennedy Shriver National Institute of Child, H., Human Development Neonatal Research, N. (2020) Early-Onset Neonatal Sepsis 2015 to 2017, the Rise of Escherichia coli, and the Need for Novel Prevention Strategies. JAMA Pediatr 174, e200593
[2] Christensen, R. D. and Rothstein, G. (1984) Pre- and postnatal development of granulocytic stem cells in the rat. Pediatr Res 18, 599–602
[3] Makoni, M., Eckert, J., Anne Pereira, H., Nizet, V., Lawrence, S. M. (2016) Alterations in neonatal neutrophil function attributable to increased immature forms. Early Hum Dev 103, 1–7
[4] Kobayashi, S. D., Voyich, J. M., Whitney, A. R., DeLeo, F. R. (2005) Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor. J Leukoc Biol 78, 1408–18.
[5] Chaudhry, H., Zhou, J., Zhong, Y., Ali, M. M., McGuire, F., Nagarkatti, P. S., Nagarkatti, M. (2013) Role of cytokines as a double-edged sword in sepsis. In Vivo 27, 669–84
[6] Mahabeleshwar, G. H., Kawanami, D., Sharma, N., Takami, Y., Zhou, G., Shi, H., Nayak, L., Jeyaraj, D., Grealy, R., White, M., McManus, R., Ryan, T., Leahy, P., Lin, Z., Haldar, S. M., Atkins, G. B., Wong, H. R., Lingrel, J. B., Jain, M. K. (2011) The myeloid transcription factor KLF2 regulates the host response to polymicrobial infection and endotoxic shock. Immunity 34, 715–28.

Tags: immune system, inflammasome, neonates, sepsis

doi: https://doi.org/10.1242/prelights.40570

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