Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20ms impulses at 200V/cm) 24h prior to the transplantation. Group A (n=5) received CsA (2.5mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123mmHg, vs 44±8mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II–IIIA). hIL-10 serum levels on day 5 were 14±7pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantation

Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20ms impulses at 200V/cm) 24h prior to the transplantation. Group A (n=5) received CsA (2.5mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123mmHg, vs 44±8mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II-IIIA). hIL-10 serum levels on day 5 were 14±7pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantatio

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

Introduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4⁺ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particle–positive CD11c⁺/CD11b⁺ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4⁺ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4⁺ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

LRR-protein RNH1 dampens the inflammasome activation and is associated with COVID-19 severity.

Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1-mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing) proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1β production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1β expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in Rnh1 <sup>-/-</sup> mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity