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Research article
Juan C. Salazar
UConn Health Center: UConn Health
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3881-546X
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Background: Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88-/- Bb-stimulated bone marrow-derived macrophages (BMDMs).
Results: MyD88-/- BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88-/- BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively.
Conclusion: Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.
Keywords
macrophage, phagocytosis, inflammation, borrelia, MyD88
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This is a list of supplementary files associated with this preprint. Click to download.
- AuthorChecklistE10only.pdf
- BetaactinCelllysates.tif
- Caspase1Celllystates.tif
- Caspase1Supernatant.tif
- IL1betaCelllystates.tif
- IL1betaSupernatant.tif
- SupplementalFile1.xlsx
- SupplementalFile2.xlsx
- SupplementalFile3.xlsx
- SupplementalFile4.xlsx
- SupplementalFile5.xlsx
- SupplementalFile6.xlsx
- FigS1.png
Correlation of reads of RNA sequencing data. (A-D) Correlation of reads of RNA isolated from Bb-stimulated BMDMs. WT BMDMs were stimulated at an MOI 10:1 for 6 hours (A). MyD88-/- BMDMs were stimulated at a MOI 100:1 for 6 hours (B). To calculate differential expression, reads from the stimulated samples were normalized to unstimulated BMDMs of the same genotype; WT (C) or MyD88-/- (D).
- FigS2.png
Secondary Control for TLR2. Confocal images of internalized Bb with AF350 Secondary Antibody in WT BMDMs after stimulation at MOI 10:1. Green is Bb, blue is AF350 Secondary Antibody, and red is actin.
- FigS3.png
MyD88-/- BMDMs stimulated with Bb show abrogated cytokine production. (A-D) Quantification of IL-6 (A), TNF (B), IL-10 (C) and CCL2 (D) proteins in supernatant from WT and MyD88-/- (MyD) BMDMs stimulated with Bb at MOI 10:1 for 1, 4 or 6 hours. N= 3 mouse BMDM per genotype *p-value<0.05, **p-value<0.01, ***p-value<0.001, NS=not significant
- FigS4.png
Bb does not induce ASC formation in BMDMs. (A-D) Confocal images (40x) of BMDMs stimulated with either Sa (A and C) or Bb (B and D) for 30 minutes (A-B) or 6 hours (C-D). Blue is nucleus, red is ASC and green is the bacteria species.
- FigS5.png
MyD88-privative MRs are significantly enriched in biological processes related to chemotaxis. (A) Venn diagram comparing biological processes (BP) relating to chemotaxis significantly enriched between MyD88-dependent (light gray) and MyD88-privative (dark gray) master regulators. (B) Heat map showing fold change of master regulators enriched in chemotaxis biological processes in MyD88-/- (yellow) BMDMs. GO numbers for significantly enriched BP are indicated on the x-axis.
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CURRENT STATUS: Under Review
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Received 15 Mar, 2021
Editorial decision: Minor revision
On 15 Mar, 2021
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Received 08 Mar, 2021
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On 23 Feb, 2021
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Reviewer #1 agreed
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This preprint is under consideration at BMC Immunology. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Juan C. Salazar
UConn Health Center: UConn Health
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3881-546X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 02 Mar, 2021
No community comments so far
Review #2 received
Received 15 Mar, 2021
Editorial decision: Minor revision
On 15 Mar, 2021
Review #1 received
Received 08 Mar, 2021
Reviewer #2 agreed
On 28 Feb, 2021
Editor assigned
On 23 Feb, 2021
Reviewers invited
Invitations sent on 23 Feb, 2021
Reviewer #1 agreed
On 23 Feb, 2021
Submission checks complete
On 23 Feb, 2021
Editor invited
On 23 Feb, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88-/- Bb-stimulated bone marrow-derived macrophages (BMDMs).
Results: MyD88-/- BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88-/- BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively.
Conclusion: Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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