For any opsonins and immune reactions it is context that determines outcome. We are addressing whether ficolins are involved in non-inflammatory homeostatic removal of pathogens (at low levels) and whether at higher levels of pathogen load the ficolins are pro-inflammatory. In addition, whether other pro-inflammatory signals such as lower pH have an effect on ficolin function.

Firstly, it must be understood that these proteins normally exist as quiescent polymers that, once activated,  can be depolymerised and consequently function to bind and modulate the response of other immune cells that encounter them; dependent on whether the ficolin is unbound or an opsonised particle and in differing pH conditions. As a recombinant molecule the L-ficolin we use for research is predominantly monomeric in format and, thus, better represents the activated (depolymerised) L-ficolin that exists in the body. 

Moreover, as unbound molecules, both head and tail binding sites are freely available and, in a manner similar to that described for SP-D, the unbound ficolin is free to mediate a pro-inflammatory response through binding of, as yet, unidentified cell surface receptors via its ‘head’. Therefore, it is not surprising that they manifest an increase in cell activation and cytokine output in our research. Moreover, the acid pH, which represents an inflammatory environment, would be another signal to the immune cells to respond in a pro-inflammatory way, thus, this would explain the increased pro-inflammatory cytokine activity we have observed.

Bound to the surface of a target the ficolins would only present their ‘tail’ to the immune system and would, thus, activate a subset of cell surface receptors that could be inhibitory, as observed with apoptotic cell removal and SP-D. As a first line of innate immune defence it is not surprising that the ficolins would seek to opsonise and remove in a non- or minimally-inflammatory way. If successful this would represent efficient removal of potential threats without large-scale inappropriate inflammation. If the pathogen is present in larger quantities this would manifest a depletion of ficolin and make available non-opsonised pathogens which would then scale up the immune response and consequently lead to pro-inflammatory cytokine production and monocyte and neutrophil recruitment to the site of infection to facilitate removal of targets. With inflammation, more serum ficolin would be made available at the site of infection, as well as more phagocytes recruited for the removal of pathogens. As the balance of free pathogen (conidia) and ficolin quantities is redressed, more of the unphagocytosed  pathogen would be opsonised by the ficolin rather than unopsonised and, thus, normal acute inflammation resolution would be facilitated, by ficolin-opsonised down regulation of pro-inflammatory cytokine production.

This is definitely an important point and it is something that we have considered. However, the purpose of the current study was to provide novel observations on the roles of L-ficolin in A. fumigatus defence. We feel that the mechanistic insights in to why L-ficolin modulates cytokines in such a manner would be best suited for a separate manuscript. It is something we are currently investigating and preliminary evidence concerning the ability of L-ficolin itself to enhance cytokine responses is linked to activation of ERK 1/2. The fact that L-ficolin can induce such a robust immune response by itself is also a surprise to us and it is not fact, but it something we can theorise on and test further. It would be interesting to investigate the mechanism further in an in vivo model whereby we could measure cytokine production in the presence of the native forms of L-ficolin and come to a more accurate conclusion. This is something we wish to investigate in the near future. 

The present data fit this profile and it is only the monomeric nature of our recombinant protein that offers some minor confusion. Our work with apoptotic cells and ficolins, which is in preparation, also fits this homeostatic model of inflammation control.