Layers of immunity: Deconstructing the Drosophila effector response

Reviewer #1 (Public review):

Summary:

The innate immune system serves as the first line of defense against invading pathogens. Four major immune-specific modules - the Toll pathway, the Imd pathway, melanization, and phagocytosis- play critical roles in orchestrating the immune response. Traditionally, most studies have focused on the function of individual modules in isolation. However, in recent years, it has become increasingly evident that effective immune defense requires intricate interactions among these pathways.

Despite this growing recognition, the precise roles, timing, and interconnections of these immune modules remain poorly understood. Moreover, addressing these questions represents a major scientific undertaking.

Strengths:

In this manuscript, Ryckebusch et al. systematically evaluate both the individual and combined contributions of these four immune modules to host defense against a range of pathogens. Their findings significantly enhance our understanding of the layered architecture of innate immunity.

Weaknesses:

While I have no critical concerns regarding the study, I do have several suggestions to offer that may help further strengthen the manuscript. These include:

(1) Have the authors validated the efficiency of the mutants used in this study? It would be helpful to include supporting data or references confirming that the mutations effectively disrupted the intended immune pathways.

(2) Given the extensive use of double, triple, and quadruple mutants, a more detailed description of the mutant construction process is warranted.

Reviewer #2 (Public review):

Summary:

In this work, the authors take a holistic view of Drosophila immunity by selecting four major components of fly immunity often studied separately (Toll signaling, Imd signaling, phagocytosis, and melanization), and studying their combinatory effects on the efficiency of the immune response. They achieve this by using fly lines mutant for one of these components, or modules, as well as for a combination of them, and testing the survival of these flies upon infection with a plethora of pathogens (bacterial, viral, and fungal).

Strengths:

It is clear that this manuscript has required a large amount of hands-on work, considering the number of pathogens, mutations, and timepoints tested. In my opinion, this work is a very welcome addition to the literature on fly immune responses, which obviously do not occur in one type of response at a time, but in parallel, subsequently, and/or are interconnected. I find that the major strength of this work is the overall concept, which is made possible by the mutations designed to target the specific immune function of each module (at least seemingly) without major effects on other functions. I believe that the combinatory mutants will be of use for the fly community and enable further studies of the interplay of these components of immune response in various settings.

To control for the effects arising from the genetic variation other than the intended mutations, the mutants have been backcrossed into a widely used, isogenized Drosophila strain called w1118. Therefore, the differences accounted for by the genotype are controlled.

I also appreciate that the authors have investigated the two possible ways of dealing with an infection: tolerance and resistance, and how the modules play into those.

Weaknesses:

While controlling for the background effects is vital, the w1118 background is problematic (an issue not limited to this manuscript) because of the wide effects of the white mutation on several phenotypes (also other than eye color/eyesight). It is a possibility that the mutation influences the functionality of the immune response components, for example, via effects of the faulty tryptophan handling on the metabolism of the animal.

I acknowledge that it is not reasonable to ask for data in different backgrounds better representing a "wild type" fly (however, that is defined is another question), but I think this matter should be brought up and discussed.

The whole study has been conducted on male flies. Immune responses show quite extensive sex-specific variation across a variety of species studied, also in the fly. But the reasons for this variation are not fully understood. Therefore, I suggest that the authors conduct a subset of experiments on female flies to see if the findings apply to both sexes, especially the infection-specificity of the module combinations.