Targeting Gα13-integrin interaction ameliorates systemic inflammation

Systemic inflammation as manifested in sepsis is an excessive, life-threatening inflammatory response to severe bacterial or viral infection or extensive injury. It is also a thrombo-inflammatory condition associated with vascular leakage/hemorrhage and thrombosis that is not effectively treated by current anti-inflammatory or anti-thrombotic drugs. Here, we show that MB2mP6 peptide nanoparticles, targeting the Gα13-mediated integrin “outside-in” signaling in leukocytes and platelets, inhibited both inflammation and thrombosis without causing hemorrhage/vascular leakage. MB2mP6 improved mouse survival when infused immediately or hours after onset of severe sepsis. Furthermore, platelet Gα13 knockout inhibited septic thrombosis whereas leukocyte Gα13 knockout diminished septic inflammation, each moderately improving survival. Dual platelet/leukocyte Gα13 knockout inhibited septic thrombosis and inflammation, further improving survival similar to MB2mP6. These results demonstrate that inflammation and thrombosis independently contribute to poor outcomes and exacerbate each other in systemic inflammation, and reveal a concept of dual anti-inflammatory/anti-thrombotic therapy without exacerbating vascular leakage.


Statistics
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Data analysis
For IHC image analysis and bacteria colony counting, ImageJ software (version 1.53e) from NIH was used. For data statistic analysis, GarphPad Prism (version 8.3.0) from GraphPad Software and Excel (office 365) from Microsoft, Inc. were used.
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Data
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
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Replication
The majority data in the study were repeated at least three times to ensure the reproducibility. Proper statistical analyses were applied to illustrate significance when it's needed. Some Key experiments were repeated by different persons in the group and all the attempts were successful.
Randomization All animals were grouped on the basis of genotypes within given age with equal amount of genders. All mice in the treatment and control groups were also randomly assigned with similar age and equal amount sexes. For ex vivo experiments, mouse platelets or bone marrow derived macrophages or neutrophils were isolated and pooled from 2-6 mice and treated as one independent experiment.

Blinding
The picture taking and quantification of IHC slides were blinded by covering the label on each slide. The CLP surgeries were performed without knowing individual genotype and the survival data were obtained and grouped by mouse ear tag with corresponding genotype record. The bacteria colony counting was blinded by calculated with different persons. In vitro experiments were not blinded because blinding are not effective in improving objectivity in these experiments and inefficient.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. 8. FITC-conjugated rat anti-mouse P-selectin antibody (553744) was used to label and detect platelet surface P-selectin expression. The product information, applications and citations can be found at: https://www.bdbiosciences.com/eu/applications/research/tcell-immunology/regulatory-t-cells/surface-markers/mouse/fitc-rat-anti-mouse-cd62p-rb4034/p/553744. 9. Rabbit anti-VWF antibody (AB7356) was used to stain VWF in IHC staining (1:50). The validation of the antibody and other product information can be found at: https://www.emdmillipore.com/US/en/product/Anti-von-Willebrand-Factor-Antibody,MM_NF-AB7356.

Wild animals
This study did not involve the use of wild animals.
Field-collected samples This study did not involve the use of field-collected samples.

Ethics oversight
Animal usage and protocol were approved by the Institutional Animal Care Committee, University of Illinois at Chicago. A randomized approach of choosing mice was used throughout the study, using all mice with the correct genotype without bias.
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