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Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages

Tatiana M. Clemente, Leonardo Augusto, Rajendra K. Angara, Stacey D. Gilk

Posted on: 16 May 2023

Preprint posted on 15 March 2023

Coxiella burnetii, the pathogen that causes Q fever, demonstrates a strategy to effectively evade the immune system by inhibiting the IL-17-induced oxidative stress in macrophages. preLight Authors: Abigail Page & Farhana Afrin Mohona

Selected by UofA IMB565

preLight Authors: Abigail Page & Farhana Afrin Mohona

Background: Coxiella burnetii is a highly infectious bacterium responsible for Q fever, a potentially fatal disease that displays flu-like symptoms or progresses into life-threatening endocarditis. The treatment for this life-threatening condition involves a two-year regimen of antibiotic combination therapy as well as valve replacement surgery. Although there is a vaccine, it is not accepted world-wide. The insufficient understanding of the bacterium’s pathogenesis imposes restrictions on these therapeutic approaches, resulting in a high mortality rate. (1-7)

Coxiella burnetii is an obligate intracellular pathogen that primarily targets alveolar macrophages. During infection, the bacterium forms phagolysosome-like vacuoles called the Coxiella-Containing Vacuole (CCV) that support their replication. Within the vacuole, the pathogen uses the Dot/Icm type IVB secretion system (T4BSS) to deliver effector proteins to control host signaling pathways and cellular processes. This T4BSS pathway helps maintain the vacuole fusogenicity, prevents host cell death, and activates inflammasomes, which are important for the immune response. But how Coxiella burnetii evades the host innate immune response is unclear.

Previous work has shown that the bacterium restricts the host immune response by blocking the signaling pathway of interleukin-17 (IL-17), a proinflammatory cytokine that aids the production of antimicrobial peptides and chemokines, leading to pathogen eradication. In this preprint, the researchers found that T4BSS downregulates the expression of IL-17 target genes and blocks IL-17-induced chemokine secretion,  protecting the pathogen from the bactericidal effect of IL-17, and promoting survival. (8-18)

In this preprint, the authors illustrate that C. burnetii T4BSS prevents the transcription pathway activated by IL-17 and disrupts the IL17RA/ACT1/TRAF6 pathway. Additionally, if the IL17R-ACT1-TRAF6 pathway is disrupted, the bactericidal effect of IL-17 is reduced.  They also show that alveolar macrophage produce high numbers of reactive oxygen species in response to IL-17, but C. burnetii T4BSS proteins completely inhibit IL-17 oxidative stress, hinting that the bacterium downregulates IL-17 signaling to avoid being killed by reactive oxygen species. (Figure 1)

Figure 1: The binding of IL-17 to the IL-17RC/IL-17RA receptor recruits ACT1, which is necessary for all known IL-17 dependant pathways. TRAF6 coordinates with ACT1 to activate NFkB, vital for the induction of proinflammatory genes. The phosphorylation of ACT1 reduces the affinity of Act1 for TRAF6, promoting TRAF2/5 interactions. This binding activates MAP kinase signaling, leading to the transcription of chemokine mRNAs. [26]

 

Key Findings:

Coxiella burnetii blocks activation of IL-17 signaling in alveolar macrophages

The researchers theorized that the T4BSS effector proteins block the ACT1/TRAF6 pathway downstream of the IL-17 transcription receptor. To test this, they conducted a colorimetric enzyme assay using HEK-Blue IL-17 cells, which stably express the IL-17RA/IL-17RC heterodimer IL-17 receptor, ACT1, and secreted embryonic alkaline phosphatase (SEAP) reporter. The authors showed that IL-17 binding to the IL-17 receptor triggers the ACT1/TRAF6 signaling cascade to activate NFkB, inducing SEAP expression. They revealed a more than 70% decrease in SEAP expression in cells infected with WT C. burnetii compared to mock and T4BSS defective mutant (ΔdotA)-infected cells. This indicated that C. burnetii inhibits the activation of the ACT1- TRAF6/ NFkB and AP-1 pathways via T4BSS effector proteins.

C. burnetii T4BSS blocks IL-17 activation of NFkB p65, MAPK, and SAPK/JNK pathways

The researchers then sought to assess whether C. burnetii targets NFkB and/or Map kinases following stimulation by IL-17 by measuring the phosphorylation levels of NFkB p-65, JNK/SAPK, and p38 MAPK in infected mouse alveolar macrophages (MH-S cells). Observed through immunoblots, the researchers observed less activation of NFkB p-65 and p38 MAPK, and a drastic reduction in JNK/SAPK activation. These findings imply that during IL-17 stimulation, C. burnetii T4BSS targets the NFkB and MAP kinase pathways, namely JNK/SAPK, as a means of preventing the transcription of IL-17 downstream genes.

Disruption of the IL17R-ACT1-TRAF6 pathway neutralizes the IL-17 bactericidal effect

The researchers sought to find out whether the IL-17R-ACT1-TRAF6 pathway is required for the bactericidal effects of IL-17. While wild type macrophage stimulated with IL-17 were able to restrict C. burnetii growth (32.7%) and reduce CCV size, knockouts of IL-17RA and TRAF6 abrogated the IL-17-mediated restriction. Depletion of ACT1 protein with siRNA resulted in recovery of bacterial growth and size of CCV which stands in contrast to the effects observed in the presence of IL-17. These contrasting outcomes strongly indicate that the IL-17-ACT1-TRAF6 pathway is essential for the bactericidal effects against Coxiella burnetii.

Coxiella burnetii T4BSS inhibits IL-17-mediated oxidative stress in macrophages

IL-17 can trigger reactive oxygen species (ROS) production by activating NADPH-oxidases (NOX) through ACT1 and TRAF6. High ROS levels inhibit growth of the bacterium. The researchers aimed to see whether Coxiella burnetii T4BSS inhibits IL-17 signaling in macrophages to avoid elevated ROS levels. They conducted a fluorometric assay and found a significant increase in NOX activity in macrophages in WT cells in the presence of IL-17, compared to untreated cells (Δil-17ra). Further, using CellROX Green, they measured ROS levels in mock, WT and ΔdotA mutant-infected cells and found a significant increase in ROS in the mutated cells and not the WT. This suggests that Coxiella burnetii employs the T4BSS to block IL-17 mediated ROS.

Conclusion:

This study shows that Coxiella burnetii targets the IL-17R-ACT1-TRAF6 pathway to inhibit transcription of IL-17 target genes and block the activity of NOX enzymes to prevent ROS, ultimately to protect themselves during immune invasion. 

Why we chose this preprint:

Q fever is a zoonotic disease caused by Coxiella burnetii that causes severe chronic illness and is transmitted through inhaling contaminated aerosols. The diagnosis and treatment of Q fever has been met with difficulty, as there is no licensed vaccine or therapy available. As researchers interested in health and medicine, this is very concerning, and this is what made this preprint so intriguing.

Essentially, Coxiella burnetii  is able to combat oxidative stress and survive in the host body. This study is vital to the understanding of the physiological relevance of how these pathogens interact with the host immune system. The authors paint a well put together picture of how this bacterium targets the IL-17R-ACT1-TRAF6 pathway. The research on how this bacterium combats oxidative stress to promote survival adds to the knowledge of how reactive oxygen stress works in the host-microbe relationship and could potentially lead to novel therapeutics. This aspect of the paper attracted us personally, as we have a great interest in understanding cell signaling mechanisms, which would enable us to better comprehend the disease prognosis and the drug mechanisms used in treating this disease. 


Questions for the Authors:

Q1. It is stated that C. burnetii has been found to use one or more T4BSS effector proteins, do you think that C. burnetii evolved to use varying numbers of T4BSS effectors to adapt to different host environments, such as when the host has an elevated or decreased immune system?

Q2. Could this process of ROS combatants be utilized for a potential therapy in battling ROS synthetically in the human body in relation to combating ROS-elevating disorders, such as cancers and chronic diseases?

Q3. What approach would be taken to discern the virulence determinants excreted by the C. burnetii T4BSS system that effectively impede the IL-17 pathway?

Q4. Do you intend to perform Real-time quantitative PCR assay for the purpose of ascertaining the quantitative levels of NFkB p65, MAPK, and SAPK/JNK subsequent to the induction of IL-17?

Q5. How might the ability of Coxiella burnetii to inhibit IL-17-mediated oxidative stress impact the development of Q fever?

References:
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16. Lin, Y., et al., Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis. Immunity, 2009. 31(5): p. 799-810.
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20. Leonardi, A., et al., CIKS, a connection to Ikappa B kinase and stress-activated protein kinase. Proc Natl Acad Sci U S A, 2000. 97(19): p. 10494-9.
21. Mahapatra, S., et al., Coxiella burnetii Employs the Dot/Icm Type IV Secretion System to Modulate Host NF-κB/RelA Activation. Front Cell Infect Microbiol, 2016. 6: p. 188.
22. Burette, M., et al., Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein. Proc Natl Acad Sci U S A, 2020. 117(24): p. 13708- 13718.
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  1. Shembade, N., Harhaj, E. IKKi: a novel regulator of Act1, IL-17 signaling and pulmonary inflammation. Cell Mol Immunol8, 447–449 (2011). https://doi.org/10.1038/cmi.2011.4

 

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