Histoplasma capsulatum Activates Hematopoietic Stem Cells and Their Progenitors through a Mechanism Dependent on TLR2, TLR4, and Dectin-1
<p>Characterization of the HSPC population. Expression of CD105<sup>+</sup> and Sca-1<sup>+</sup> surface antigens on mouse bone marrow long-term culture initiator cells (LT-CIC or LT-HSC). (<b>A</b>) Control of Lin<sup>−</sup> cells without positive selection; (<b>B</b>) CD105<sup>+</sup> Sca-1<sup>+</sup> cells corresponding to HSPC.</p> "> Figure 2
<p>Expression of TLR2, TLR4 and Dectin-1 receptors in HSPC. (<b>A</b>,<b>D</b>) TLR2 expression in unstimulated and stimulated HSPC with <span class="html-italic">H. capsulatum</span> yeasts; (<b>B</b>,<b>E</b>) TLR4 expression in unstimulated and stimulated HSPC with <span class="html-italic">H. capsulatum</span> yeasts; (<b>C</b>,<b>F</b>) Expression of Dectin-1 in unstimulated and stimulated HSPC with <span class="html-italic">H. capsulatum</span> yeasts. HSPCs were selected from the CD105<sup>+</sup>/Sca-1<sup>+</sup> population and the level of expression of receptors is expressed as mean fluorescence intensity (MFI). (<b>A</b>–<b>D</b>) Results are expressed as means ± SD of pooled data from three independent experiments. (<b>D</b>–<b>F</b>) Data represent the percentage of HSPC positive cells and are from a representative experiment of three replicates, ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 3
<p>Phagocytosis of <span class="html-italic">H. capsulatum</span> yeasts by HSPC. Phagocytosis was analyzed by flow cytometry and the result is expressed as the percentage of FITC positive cells (% phagocytosis) of HSPC cells infected with <span class="html-italic">H. capsulatum</span> yeasts. (<b>A</b>) Control, HSPC (<b>B</b>) Control, percentage of phagocytosis in HSPC co-cultured with <span class="html-italic">H. capsulatum</span> and without treatment; (<b>C</b>) percentage of phagocytosis in HSPC treated with anti-TLR2; (<b>D</b>) percentage of phagocytosis in HSPC treated with anti-TLR4; (<b>E</b>) percentage of phagocytosis in HSPC treated with the peptide CLEC7A; (<b>F</b>) percentage of phagocytosis in HSPC treated with the anti-TLR2/anti-TLR4 combination; (<b>G</b>) percentage of phagocytosis in HSPC treated with the anti-TLR2/CLEC7A combination; (<b>H</b>) percentage of phagocytosis in HSPC treated with the combination of anti-TLR4/CLEC7A, and (<b>I</b>) percentage of phagocytosis in HSPC treated with the combination of anti-TLR2/anti-TLR4/CLEC7A. Histograms in gray correspond to HSPC previously treated with blocking antibodies for TLR or with a specific peptide for Dectin-1 (CLEC7A). Data are representative from an experiment of three replicates; *** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 4
<p>Fungicidal activity of HSPC against <span class="html-italic">H. capsulatum</span>. Colony forming units (CFUs) were recovered from HPSC infected with <span class="html-italic">H. capsulatum</span> yeasts after incubation for 24 h at 37 °C. Results are expressed as median and IQR of pooled data from three independent experiments.</p> "> Figure 5
<p>Expression of cytokines and inflammatory mediators in HSPC stimulated with <span class="html-italic">H. capsulatum</span> yeast. Analysis of mRNA expression of proinflammatory cytokines, arginase-1, and iNOS in HSPC stimulated or not stimulated with <span class="html-italic">H. capsulatum</span> yeast. (<b>A</b>) IL-6; (<b>B</b>) IL-17; (<b>C</b>) IL-1β; (<b>D</b>) IL-10; (<b>E</b>) TNF-α; (<b>F</b>) TGF-β1; (<b>G</b>) Arg-1; and (<b>H</b>) iNOS. HSPC, control, unstimulated cells; HSPC + Hc, cells stimulated with <span class="html-italic">H. capsulatum</span>; TLR, <span class="html-italic">Toll</span>-like receptor; CLEC7A, peptide blocker specific for Dectin-1. Results are expressed as means ± SD of pooled data from three independent experiments; * <span class="html-italic">p</span> < 0.0001, comparisons were done between HSPCs + <span class="html-italic">Hc</span> vs. HSPCs, and <sup>#</sup> <span class="html-italic">p</span> < 0.0001 HSPCs + <span class="html-italic">Hc</span> plus the different treatments vs. HSPCs + <span class="html-italic">Hc</span>.</p> "> Figure 6
<p><span class="html-italic">Histoplasma capsulatum</span> induces apoptosis and necrosis in HSPC. HSPCs were treated with Annexin V-FITC and propidium iodide as described in materials and methods. (<b>A</b>) Control, uninfected HSPC; (<b>B</b>) HSPC stimulated with <span class="html-italic">H. capsulatum</span> yeasts. Percentages represent the number of cells positive for FITC and propidium iodide. Similar results were obtained from three independent experiments.</p> "> Figure 7
<p><span class="html-italic">H. capsulatum</span> yeasts affect HSPC proliferation. (<b>A</b>) Control, unstimulated HSPC; (<b>B</b>) HSPC + Pam3CysOH; (<b>C</b>) HSPC + LPS; <b>(D</b>) HSPC + β-glucan; (<b>E</b>) HSPC + Pam3CysOH + <span class="html-italic">H. capsulatum</span>; (<b>F</b>) HSPC + LPS + <span class="html-italic">H. capsulatum</span>; (<b>G</b>) HSPC + β-glucan + <span class="html-italic">H. capsulatum</span>. Data represent the percentage of BrdU positive cells; results are from a representative experiment of three replicates.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethical Considerations
2.2. Isolation, Purification, and Maintenance of Bone Marrow-Derived HSPCs
2.3. Determination of the TLR2, TLR4, and Dectin-1 Expression on HSPC
2.4. Histoplasma Capsulatum Yeasts
2.5. Culture of HSPC with H. capsulatum Yeasts
2.6. Phagocytosis Assay
2.7. Microbicidal Activity of HSPCs against H. capsulatum Yeasts
2.8. Determination of the Expression of Cytokines and Inflammatory Mediators by Real-Time Quantitative PCR (qPCR)
2.9. Apoptosis Assay
2.10. Cell Proliferation Assay
2.11. Statistical Analysis
3. Results
3.1. Identification of the Bone Marrow-Derived HSPC Population
3.2. Histoplasma Capsulatum induces Increased Expression of TLR2 and Dectin-1 in HSPC
3.3. HSPCs Are Able to Phagocytose H. capsulatum Yeasts but Do Not Exert a Fungicidal Effect
3.4. Histoplasma Capsulatum induces the Expression of Cytokines and Inflammatory Mediators in HSPCs
3.5. H. capsulatum Yeasts Induce Apoptosis and Necrosis and Affect Proliferation in HSPCs
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Forward (5′-3′) | Reverse (5′-3′) |
---|---|---|
GAPDH | CATGGCCTTCCGTGTTCCTA | GCGGCACGTCAGATCCA |
IL-6 | CAACCACGGCCTTCCCTACTTC | TCTCATTTCCACGATTTCCCAGAG |
IL-17 | CCAAACACTGAGGCCAAGGACTTC | GGTGACGTGGAACGGTTGAGGTAG |
IL-1β | CTTCAAATCTCGCAGCAGCACATC | TCCACGGGAAAGACACAGGTAGC |
IL-10 | TGGGTTGCCAAGCCTTATCGG | CTCACCCAGGGAATTCAAATGCTC |
TNF-α | GACAAGGCTGCCCCGACTACG | CTTGGGGCAGGGGCTCTTGAC |
TGFβ | TACTGCCGCTTCTGCTCCCACTCC | TCGATGCGCTTCCGTTTCACCAG |
Arg-1 | CCTTGGCTTGCTTCGGAACTCA | CTTGGGAGGAGAAGGCGTTTGC |
iNOS | GCCGCATGAGCTTGGTGTTTG | GCAGCCGGGAGTAGCCTGTGT |
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Rodríguez-Echeverri, C.; Gómez, B.L.; González, Á. Histoplasma capsulatum Activates Hematopoietic Stem Cells and Their Progenitors through a Mechanism Dependent on TLR2, TLR4, and Dectin-1. J. Fungi 2022, 8, 1108. https://doi.org/10.3390/jof8101108
Rodríguez-Echeverri C, Gómez BL, González Á. Histoplasma capsulatum Activates Hematopoietic Stem Cells and Their Progenitors through a Mechanism Dependent on TLR2, TLR4, and Dectin-1. Journal of Fungi. 2022; 8(10):1108. https://doi.org/10.3390/jof8101108
Chicago/Turabian StyleRodríguez-Echeverri, Carolina, Beatriz L. Gómez, and Ángel González. 2022. "Histoplasma capsulatum Activates Hematopoietic Stem Cells and Their Progenitors through a Mechanism Dependent on TLR2, TLR4, and Dectin-1" Journal of Fungi 8, no. 10: 1108. https://doi.org/10.3390/jof8101108
APA StyleRodríguez-Echeverri, C., Gómez, B. L., & González, Á. (2022). Histoplasma capsulatum Activates Hematopoietic Stem Cells and Their Progenitors through a Mechanism Dependent on TLR2, TLR4, and Dectin-1. Journal of Fungi, 8(10), 1108. https://doi.org/10.3390/jof8101108