The protease content in HDM is responsible for the IgE but not the IgG1 response.

<p>Allergic sensitization response after HDM exposure in Wt and Par-2 deficient mice. Total IgE, HDM-specific IgE and HDM-specific IgG1 ELISA measurements were performed in serum collected 24 hours after the last PBS/HDM exposure. Measurements of total IgE after PBS and (A) HDM Greer exposure or (B) HDM Citeq exposure, HDM-IgE after PBS and (C) HDM Greer exposure or (D) HDM Citeq exposure, HDM-IgG1 after PBS and (E) HDM Greer exposure or (F) HDM Citeq exposure. Median levels are shown (n = 6–8 mice per group). *p<0.05, **p<0.01 and ***p<0.001 between PBS and HDM exposed mice or between Wt and Par-2 deficient mice.</p

Par-2-deficiency does not influence the inflammatory response after HDM exposure.

<p>Total cell counts and mononuclear, eosinophil and neutrophil fractions in BALF from Wt or Par-2 deficient mice after chronic exposure to PBS or HDM (Greer/Citeq). BALF cells were counted 24 hours after the last PBS/HDM exposure. Total cell count after PBS and (A) HDM Greer exposure or (B) HDM Citeq exposure, Mononuclear cell count (%) after PBS and (C) HDM Greer exposure or (D) HDM Citeq exposure, Eosinophil cell count (%) after PBS and (E) HDM Greer exposure or (F) HDM Citeq exposure, Neutrophil cell count (%) after PBS and (G) HDM Greer exposure or (H) HDM Citeq exposure. Median levels are shown (n = 7–8 mice per group). *p<0.05 and **p<0.01 between PBS and HDM exposed mice or differences between Wt and Par-2 deficient mice.</p

Par-2 deficiency affects IL-17 but not KC or IFNγ production after HDM exposure.

<p>ELISA measurements were performed in homogenized lung lysates from lungs collected 24/HDM exposure. Measurements of KC after PBS and (A) HDM Greer exposure or (B) HDM Citeq exposure, IL-17 after PBS and (C) HDM Greer exposure or (D) HDM Citeq exposure, and IFNγ after PBS and (E) HDM Greer exposure or (F) HDM Citeq exposure. Median levels are shown (n = 6–8 mice per group). *p<0.05 and **p<0.01 between PBS and HDM exposed mice.</p

ROS and GSH levels at basal levels and 24 hours after 4Gy of irradiation.

<p><b>A.</b> ROS levels expressed in mean intensity at baseline and 24 hours after irradiation, relative to each parental celline at baseline conditions. <b>B.</b> Ratio of GSH:GSSG levels at baseline and at 24 hours after irradiation for parental and ρ⁰ cell lines. Data represents the mean ± SEM from at least 3 independent biological repeats. mtDNA depleted cells are indicated by the dashed bars. * p<0.05. <b>C.</b> mRNA expression of KEAP1 and SOD2 24 hours after irradiation, normalized to each parental at baseline. Data represents the mean + SEM from at least 2 independent biological repeats. * p<0.05.</p

Distinct radiation responses after <i>in vitro</i> mtDNA depletion are potentially related to oxidative stress

<div><p>Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ⁰). Overall survival after irradiation was increased (p<0.001) for BEAS-2B ρ⁰ cells, while decreased for both tumor ρ⁰ lines (p<0.05). In agreement, increased residual DNA damage was observed after mtDNA depletion for A549 and 143B cells. Intrinsic radiosensitivity (surviving fraction at 2Gy) was not influenced. We investigated whether ROS levels, oxidative stress and/or antioxidant responses were responsible for altered radiation responses. Baseline ROS formation was similar between BEAS-2B parental and ρ⁰ cells, while reduced in A549 and 143B ρ⁰ cells, compared to their parental counterparts. After irradiation, ROS levels significantly increased for all parental cell lines, while levels for ρ⁰ cells remained unchanged. In order to investigate the presence of oxidative stress upon irradiation reduced glutathione: oxidized glutathione (GSH:GSSG) ratios were determined. Irradiation reduced GSH:GSSG ratios for BEAS-2B parental and 143B ρ⁰, while for A549 this ratio remained equal. Additionally, changes in antioxidant responses were observed. Our results indicate that mtDNA depletion results in varying radiation responses potentially involving variations in cellular ROS and antioxidant defence mechanisms. We therefore suggest when mitotoxic drugs are combined with radiation, in particular at high dose per fraction, the effect of these drugs on mtDNA copy number should be explored.</p></div

Radiation response in mtDNA depleted cell lines.

<p><b>A.</b> Clonogenic survival plots, fitted according to the LQ model. Results show mean ± SEM from at least 3 independent biological replicates. 0 Gy conditions are sham irradiated. <b>B.</b> Representative merged fluorescent images of γH2AX foci (green) and nuclei (blue) visualizing residual foci upon irradiation.</p

Validation of mtDNA depleted cell lines.

<p><b>A.</b> qPCR assessed mtDNA copy number levels expressed in percentage normalized to each parental (left panel) and the doubling time for all investigated cell lines (right panel). <b>B.</b> Basal respiration expressed as oxygen consumption rate (OCR) in function of time (minutes). <b>C.</b> Stacked plot of mitochondrial (left panel) or glycolysis (right panel) stress test after measuring OCR or extracellular acidification rate (ECAR) respectively. <b>D.</b> Relative ATP levels in A.U. corrected for cell number (left panel) and L-Lactic acid levels in g/l corrected for cell number (right panel). Data represent the mean ± SEM from at least 3 independent biological experiments. * p<0.05, *** p<0.001.</p