Complement component C1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury

Hypoxic–ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia–ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia–ischemia. Only C1q−/− mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q−/− brain mitochondria and preserved activity of the respiratory chain. Compared with C1q+/+ neurons, cortical C1q−/− neurons exhibited resistance to oxygen–glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q−/− neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation

Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59

The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria

C3^−/− mDCs display similar capacity for virus uptake and maturation.

<p>(<b>A</b>) Dot plots show the percentage of DiD flu⁺ uptake by CD103⁺ DC (left panel) and CD11b⁺ DC (right panel) in WT and C3^−/− mice 16 hrs after DiD flu administration on day 1 post infection with PR8. (<b>B</b>) Bar graph shows the average numbers of DiD flu⁺ CD103⁺ DCs and DiD flu⁺ CD11b⁺ DCs in the lungs of WT and C3^−/− mice. (<b>C</b>)Single cell preparations from the lungs were enriched for DCs by a density gradient method. Enriched DCs were infected with influenza virus under <i>ex vivo</i> culture conditions and six hours later the CD103⁺ and CD11b⁺ DCs were flow sorted. Q-RT-PCR for the indicated cytokines were performed on the RNA obtained from the CD103⁺ and CD11b⁺ DC populations. Bar graph shows % relative expression in comparison to un-infected control. (<b>D</b>) WT and C3^−/− mice were infected with flu and 24 hours later the CD103⁺ and CD11b⁺ DCs from the lungs were flow sorted. Q-RT-PCR for the indicated cytokines were performed on the RNA obtained from the CD103⁺ and CD11b⁺ DC populations. Bar graph shows % relative expression in comparison to un-infected control. (<b>E</b>) Bar graphs shows the mean fluorescent intensity of CD86, CD40 and CD80 expression on CD103⁺DC and CD11b⁺ DC subsets in the lungs of naïve and PR8 infected WT or C3^−/− mice. The data are representative of three different experiments with similar results. The values are expressed as mean ± SEM.</p

C3^−/− mice show greater weight loss and mortality during influenza infection.

<p>(<b>A</b>) Percentage of body weight loss after influenza infection. A weight loss of <20% and recovery represents a sub-lethal infection. (<b>B</b>) Survival curve comparing WT and C3^−/− mice during influenza infection (n = 6–7 in each group). (<b>C</b>) Plots represent <i>ex vivo</i> analysis of CFSE-labeled OT-I CD8⁺ T cell proliferation in the dLN 3 days after infection with PR8 and PR8-OT-I (bottom). The graph on the right shows the respective division index for the proliferating OT-I CD8⁺ T cells for each group of mice. (<b>D</b>) Plots represent <i>ex vivo</i> analysis of CFSE-labeled OT-II CD4⁺ T cell proliferation in the dLN 3 days after infection with PR8 and PR8-OT-II (bottom). The graph on the right shows the respective division index for the proliferating OT-II CD4⁺ T cells for each group of mice. (<b>E</b>) Histogram showing expression of costimulatory molecules CD86, CD80 and CD40 on CD103⁺ DCs and CD11b⁺ DCs in the dLN on day 2 post infection. Filled histogram: Isotype control, dashed histogram: Naïve, open histogram: infected. (<b>F</b>) Bar graphs show the mean fluorescent intensity (MFI) of CD86, CD80 and CD40 expression on CD103⁺DC and CD11b⁺ DC subsets in the dLN of naïve and influenza infected (Day 2) WT or C3^−/− mice. (<b>G</b>) Plots represent CFSE-labeled OT-I CD8⁺ T cell proliferation 3 days after co-culture with sorted CD103⁺ DCs and CD11b⁺ DCs obtained from pooled dLN of PR8-OT-I influenza infected mice at a ratio of 1∶10 (DC:T cells). Results shown are representative of four experiments with similar results. The values are expressed as mean ± SEM.</p

Langerin-DTR mice show effector T cells response and survival characteristics similar to those of C3^−/− mice upon influenza infection and has defective CD11b⁺ DC migration.

<p>(<b>A</b>) Plots represent <i>ex vivo</i> analysis of CFSE-labeled OT-I CD8⁺ T cells proliferation in the dLN 3 days after infection with PR8 and PR8-OT-I (bottom) in WT and CD103⁺ DCs depleted langerin-DTR mice. (<b>B</b>) Percentage of body weight loss after influenza infection. (<b>C</b>) Survival curve comparing WT, C3^−/− and Langerin –DTR mice during influenza infection (n = 6 per group). (<b>D</b>) Graph shows the relative mRNA expression level of influenza M protein in lung tissues of WT and CD103⁺ DCs depleted langerin-DTR mice infected with 4 PFU of PR8. (<b>E</b>) Graph shows the frequency of IFNγ secreting CD4⁺ T cells in lungs by <i>ex vivo</i> overnight stimulation with MHC-II flu peptide on day 7 post infection. (<b>F</b>) Bar graph shows the absolute numbers of IFNγ secreting CD4⁺ T cells in lungs on day 7 post infection (<b>G</b>) Graph shows the frequency of Flu specific CTL response in lung as measured by Flu _peptide (ASNENMETM (NP _366–374)/H-2D^b tetramer staining on day 7 post infection. (<b>H</b>) Bar graph shows the absolute numbers of flu specific CD8⁺ T cells in lungs by tetramer staining on day 7 post infection. DT treated WT and Langerin-DTR mice were flu infected and the migration of CD103⁺ and CD11b⁺ DCs were evaluated as described before on day 3 post infection. (<b>I</b>) Dot plots show CD103⁺ and CD11b⁺ DCs mDCs in the dLN of DT treated and untreated WT and Langerin-DTR mice. Numbers within the dot plot represent cell number. (<b>J</b>) Total number of CD103⁺(top) and CD11b⁺(bottom) DCs in the dLN of flu infected mice. Results shown are representative of at least three different experiments with similar results. The values are expressed as mean ± SEM.</p