Acute Complement Inhibition Potentiates Neurorehabilitation and Enhances tPA-Mediated Neuroprotection.

Because complement activation in the subacute or chronic phase after stroke was recently shown to stimulate neural plasticity, we investigated how complement activation and complement inhibition in the acute phase after murine stroke interacts with subsequent rehabilitation therapy to modulate neuroinflammation and neural remodeling. We additionally investigated how complement and complement inhibition interacts with tissue plasminogen activator (tPA), the other standard of care therapy for stroke, and a U.S. Food and Drug Administration preclinical requirement for translation of an experimental stroke therapy. CR2fH, an injury site-targeted inhibitor of the alternative complement pathway, significantly reduced infarct volume, hemorrhagic transformation, and mortality and significantly improved long-term motor and cognitive performance when administered 1.5 or 24 h after middle cerebral artery occlusion. CR2fH interrupted a poststroke inflammatory process and significantly reduced inflammatory cytokine release, microglial activation, and astrocytosis. Rehabilitation alone showed mild anti-inflammatory effects, including reduced complement activation, but only improved cognitive recovery. CR2fH combined with rehabilitation significantly potentiated cognitive and motor recovery compared with either intervention alone and was associated with higher growth factor release and enhanced rehabilitation-induced neuroblast migration and axonal remodeling. Similar outcomes were seen in adult, aged, and female mice. Using a microembolic model, CR2fH administered in combination with acute tPA therapy improved overall survival and enhanced the neuroprotective effects of tPA, extending the treatment window for tPA therapy. A human counterpart of CR2fH has been shown to be safe and nonimmunogenic in humans and we have demonstrated robust deposition of C3d, the CR2fH targeting epitope, in ischemic human brains after stroke.SIGNIFICANCE STATEMENT Complement inhibition is a potential therapeutic approach for stroke, but it is not known how complement inhibition would interact with current standards of care. We show that, after murine ischemic stroke, rehabilitation alone induced mild anti-inflammatory effects and improved cognitive, but not motor recovery. However, brain-targeted and specific inhibition of the alternative complement pathway, when combined with rehabilitation, significantly potentiated cognitive and motor recovery compared with either intervention alone via mechanisms involving neuroregeneration and enhanced brain remodeling. Further, inhibiting the alternative pathway of complement significantly enhanced the neuroprotective effects of thrombolytic therapy and markedly expanded the therapeutic window for thrombolytic therapy.

Complex Ecotype Dynamics Evolve in Response to Fluctuating Resources.

Ecotypic diversification and its associated cooperative behaviors are frequently observed in natural microbial populations whose access to resources is often sporadic. However, the extent to which fluctuations in resource availability influence the emergence of cooperative ecotypes is not fully understood. To determine how exposure to repeated resource limitation affects the establishment and long-term maintenance of ecotypes in a structured environment, we followed 32 populations of Escherichia coli evolving to either 1-day or 10-day feast/famine cycles for 900 days. Population-level analysis revealed that compared to populations evolving to 1-day cycles, 10-day populations evolved increased biofilm density, higher parallelism in mutational targets, and increased mutation rates. As previous investigations of evolution in structured environments have identified biofilm formation as the earliest observable phenotype associated with diversification of ecotypes, we revived cultures midway through the evolutionary process and conducted additional genomic, transcriptional, and phenotypic analyses of clones isolated from these evolving populations. We found not only that 10-day feast/famine cycles support multiple ecotypes but also that these ecotypes exhibit cooperative behavior. Consistent with the black queen hypothesis, or evolution of cooperation by gene loss, transcriptomic evidence suggests the evolution of bidirectional cross-feeding behaviors based on essential resources. These results provide insight into how analogous cooperative relationships may emerge in natural microbial communities. IMPORTANCE Despite regular feast and famine conditions representing an environmental pressure that is commonly encountered by microbial communities, the evolutionary outcomes of repeated cycles of feast and famine have been less studied. By experimentally evolving initially isogenic Escherichia coli populations to 10-day feast/famine cycles, we observed rapid diversification into ecotypes with evidence of bidirectional cross-feeding on costly resources and frequency-dependent fitness. Although unidirectional cross-feeding has been repeatedly observed to evolve in laboratory culture, most investigations of bidirectional cooperative behaviors in microbial populations have been conducted in engineered communities. This work demonstrates the de novo evolution of black queen relationships in a microbial population originating from a single ancestor, providing a model for investigation of the eco-evolutionary processes leading to mutualistic cooperation.