CRISPR-Cas9–mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate–induced inflammation in bovine mammary epithelial cells

Shandilya, Umesh K.; Sharma, Ankita; Mallikarjunappa, S.; Guo, Jian-Chun; Jiang, Mao Yong; Meade, Kieran G.; Karrow, Niel A.

doi:10.3168/jds.2021-20305

Cited by 11 publications

(14 citation statements)

References 51 publications

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“…MSCs express functional TLR4 34,35 , and here we have shown that activated TLR4 is localized intracellularly. MSCs can be polarized into a pro-inflammatory and anti-inflammatory phenotype by ligands of TLRs, leading to respective changes in their immunomodulatory and reparative properties [36][37][38][39] .…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy

Schary

Rotem

Caller

et al. 2023

Sci Rep

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Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.

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Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy

Schary

Rotem

Caller

et al. 2023

Sci Rep

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“…Sharp, clear 28S and 18S rRNA bands with their ratio about 2.0 were obtained on a denaturing gel indicated intact total RNA. As previously described ( Shandilya et al., 2021 ), the purified RNA samples were reverse-transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Then qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in a total reaction volume of 10 μL in a PCR 96-well plate (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

“…The polymerase was activated at 95 °C for 10 min and the PCR was performed for 40 cycles (95 °C for 15 s and 60 °C for 1 min) using a StepOne Plus instrument (Applied Biosystems). The primers were either selected based on previous studies ( Lee et al., 2019 ; Shandilya et al., 2021 ; Wu et al., 2022 ; Zhao et al., 2016 ) or designed using Primer3Plus software ( Hung and Weng, 2016 ; Mallikarjunappa et al., 2020 ). The sequences of the selected primers are listed in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

“…The sequences of the selected primers are listed in Table 1 . Primer specificity was checked by sequence alignment using Nucleotide BLAST at the NCBI genome browser gateway, and was further confirmed in a standard PCR reaction followed by ethidium-bromide staining on 2% agarose gel as previously described ( Shandilya et al., 2021 ). The reference genes glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) ( Sharma et al., 2019 ) and ubiquitously expressed prefoldin like chaperone ( UXT ) ( Wang et al., 2022 ) were used as the internal control to normalize the expression of the target gene transcript levels.…”

Section: Methodsmentioning

confidence: 99%

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Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function

Xu¹,

Shandilya²,

Yiannikouris³

et al. 2023

Animal Nutrition

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“…MSCs can be polarized into a proinflammatory and anti-inflammatory phenotype by ligands of TLRs, leading to respective changes in their immunomodulatory and reparative properties. [43][44][45][46] We have shown that TLR4 deficiency in human MSCs improves cell survival and LV function after MI in mice.…”

Section: Targeting Tlr4 To Enhance the Therapeutic Properties Of Hmscsmentioning

confidence: 99%

CRISPR-Cas9 Editing of TLR4 to Improve the Outcome of Cardiac Cell Therapy

Schary

Rotem

Caller

et al. 2022

Preprint

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Background: Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. Methods and results: We developed and optimized a new electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles, pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular (LV) remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. Conclusions: We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs, and improves infarct healing and remodeling in mice. Our results provide a new approach to improve the outcomes of cell therapy for cardiovascular diseases.

show abstract

CRISPR-Cas9–mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate–induced inflammation in bovine mammary epithelial cells

Cited by 11 publications

References 51 publications

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function

CRISPR-Cas9 Editing of TLR4 to Improve the Outcome of Cardiac Cell Therapy

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