Description
Cellular immune responses are an important aspect of innate host defense against infection and are broadly conserved from insects to mammals. The model organism
Drosophila melanogaster
uses the cellular encapsulation response to protect against macroparasite infection (Carton
et al.
, 2008; Mortimer, 2013). This response shows genetic conservation with human immune responses (Howell
et al.
, 2012), and may serve as a useful model to better understand human immune cell functions.
Drosophila
larvae are commonly infected by parasitoid wasps and following infection mount a cellular immune response to kill the parasite. This response is mediated by two cell types, circulating macrophage-like immune cells known as plasmatocytes and infection-induced immune cells called lamellocytes (Honti
et al.
, 2014; Rizki, 1957). Plasmatocytes operate as the first line responders to infection by recognizing and binding to the wasp egg (Mortimer
et al.
, 2012; Russo
et al.
, 1996). This process is then followed by the production of lamellocytes that form a consolidated multi-layered capsule, thereby killing the wasp (Kim-Jo
et al.
, 2019; Russo
et al.
, 1996). Recent findings have begun to elucidate the regulation of the encapsulation response in
Drosophila
, including a role for the evolutionarily conserved JAK-STAT signaling pathway (Sorrentino
et al.
2004; Yang
et al.
2015). The roles of JAK-STAT signaling are not completely understood, but the pathway has been linked to the production of lamellocytes (Bausek and Zeidler, 2014; Hanratty and Dearolf, 1993; Luo
et al.
, 1995, 1997; Sorrentino
et al.
, 2004).
Members of the S1A protease family are involved in many physiological processes, including the regulation of invertebrate immune responses (Cao and Jiang, 2018). In
Drosophila
,
the S1A family is composed of more than 200 genes and includes the catalytically active serine proteases (SPs) and the serine protease homologs (SPHs), a group of SP-like proteins that are enzymatically inactive (Cao and Jiang, 2018). Many S1A family members have been linked to the antimicrobial immune response including the SP genes
spirit
,
grass
,
psh
and
SPE,
and the SPH genes
sphe, sphinx1
and
sphinx2
(Buchon
et al.
, 2009; El Chamy
et al.
, 2008; Kambris
et al.
, 2006; Ligoxygakis
et al.
, 2002; Patrnogic and Leclerc, 2017). However, the role of SP and SPH genes in regulating the fly antiparasitoid immune response is still not well-defined.
A recent study of transcriptional targets of JAK-STAT pathway activity showed that the S1A family members, the SP gene
CG10764
(also known as SP77) and the SPH gene
CG4793
(also known as cSPH128) are JAK-STAT pathway target genes (Bina
et al.
, 2010). The JAK-STAT pathway is important for the production of lamellocytes following parasitoid infection (Sorrentino
et al.
, 2004; Yang
et al.
, 2015), and ectopic pathway activity leads to tumorigenesis as characterized by the precocious accumulation of lamellocytes (Ekas
et al.
, 2010; Harrison
et al.
, 1995). RNA interference (RNAi) mediated knock down of
CG10764
and
CG4793
in the JAK-STAT tumor model suggested that these genes may play antagonistic roles in regulating JAK-STAT signaling and lamellocyte production (Bina
et al.
, 2010).
To evaluate the functional roles of these JAK-STAT regulated S1A family members in fly cellular immunity, we used two different RNAi lines with unique sequence targets to knock down each gene in both the plasmatocyte (using
eater-GAL4
) (Tokusumi
et al.
2009a) and lamellocyte (using
msn-GAL4
) (Lam,
et al.
2010; Tokusumi
et al.
2009b) immune cell types and compared their ability to encapsulate parasitoid wasp eggs following infection. We find that knocking down
CG10764
with either of the RNAi lines in lamellocytes (Figure 1A;
UAS-CG10764
GL01210
: p= 0.00222, n
EXP
= 73, n
CTRL
= 72;
UAS-CG10764
NIG.10764R
: p=0.0112, n
EXP
= 71 , n
CTRL
= 67) or plasmatocytes (Figure 1B;
UAS-CG10764
GL01210
: p= 0.000955, n
EXP
= 75, n
CTRL
= 81 ;
UAS-CG10764
NIG.10764R
: p= 1.74e-06, n
EXP
= 57 , n
CTRL
= 62) results in a significant reduction in the proportion of wasp eggs that are successfully encapsulated. These findings suggest that
CG10764
may act as a positive regulator of encapsulation in both fly immune cell types. Conversely, RNAi-mediated knock down of
CG4793
in lamellocytes with either of the RNAi lines results in a significant increase in encapsulation rate (Figure 1A;
UAS-CG4793
HMC03765
: p= 1.09e- 05, n
EXP,
= 80, n
CTRL
= 72 ;
UAS-
CG4793
NIG.4793R
: p= 0.0098, n
EXP,
= 60, n
CTRL
= 67), but has no effect when knocked down in plasmatocytes (Figure 1B;
UAS-CG4793
HMC03765
: p= 0.56975, n
EXP,
=57, n
CTRL
=62 ;
UAS-
CG4793
NIG.4793R
: p= 0.321 , n
EXP,
= 57 , n
CTRL
= 62). This suggests that
CG4793
may act as a negative regulator of encapsulation specifically in lamellocytes, the immune cell subtype that is induced following infection.
Based on our observations, we hypothesize that
CG10764
and
CG4793
play important and distinct roles in balancing immune activation.
CG10764
appears to regulate the initiation of pro-immune signaling which triggers the host immune response against parasitoid infection.
CG10764
likely encodes an active serine protease, and may influence immune activation through the direct cleavage of target proteins. On the other hand,
CG4793
appears to be responsible for limiting the immune response when the defense mechanism is elicited. This is an important role which allows the host to avoid self-directed immune damage due to an overreactive immune system.
CG4793
is an SPH gene and encodes a protein that is predicted to be catalytically inactive. However, these SPH proteins play regulatory roles in a variety of processes (Cao and Jiang, 2018), and it is likely that
CG4793
is acting through a similar mechanism to limit immune activity. Thus, these S1A family members likely have cell-specific roles and regulate the cellular encapsulation process through distinct mechanisms.
A role for
CG10764
and
CG4793
in modulating JAK-STAT pathway activity has been previously demonstrated (Bina
et al.
, 2010). Interestingly, these S1A family members were also shown to have opposing effects on the phenotype seen in
hop
Tum
flies, which display a melanotic phenotype due to ectopic JAK-STAT signaling (Bina
et al.
, 2010; Hanratty and Dearolf, 1993; Luo
et al.
, 1995). Here we show that
CG10764
and
CG4793
may also act antagonistically to maintain a balanced immune response and based on these previous studies, we hypothesize that this could potentially be via regulation of JAK-STAT signaling. However, a detailed mechanistic understanding of how these S1A family genes regulate cellular immunity and how their activity may be linked to JAK-STAT pathway signaling remain to be established. Additionally, further research into the human homologs of
CG10764
and
CG4793
may reveal conserved functions in human immunity and JAK-STAT mediated disease.
Methods
Drosophila
genetics.
Tissue-specific modulation of gene expression can be achieved in
D. melanogaster
using the yeast-derived UAS-GAL4 system. GAL4 is a transcription factor that binds to the UAS enhancer sequence present in the promoter region controlling expression of the gene of interest (Brand and Perrimon, 1993). We used hemocyte specific
GAL4
lines and two
UAS-RNAi
lines with distinct target sequences to knock down the genes of interest in each hemocyte type.
UAS-GAL4
RNAi
was used as the control genotype. Independent control experiments were run with each
UAS-RNAi
experiment;
UAS-GAL4
RNAi
-1 refers to the control replicates for experiments with the
UAS-CG4793
HMC03765
and
UAS-CG10764
GL01210
constructs and
UAS-GAL4
RNAi
-2 refers to the control replicates for experiments with the
UAS-CG10764
NIG.10764R
and
UAS-
CG4793
NIG.4793R
constructs. All
Drosophila
crosses were maintained on standard
Drosophila
medium (Molasses Formulation, Genesee Scientific) at 25C° on a 12 hour light:dark cycle.
Parasitoid wasp infection.
For each genotype tested, approximately 25 virgin female
GAL4
flies were mated with 10
UAS-RNAi
line males. These crosses were transferred to egg lay chambers containing grape-juice plates (Genesee Scientific) supplemented with yeast paste and allowed to lay for 72 hours. For infection experiments, 25 F
1
second instar larvae were picked from the egg lay plates and transferred into small petri dishes with standard
Drosophila
medium (Molasses Formulation, Genesee Scientific) together with 3 female LcNet wasps. All of the surviving larvae (~25/infection plate) were dissected 72 hours post infection and the number of encapsulated wasp eggs and live wasp larvae were counted. Each genotype for each experiment was performed in triplicate. All experimental crosses and infections were carried out at 25°C.
Encapsulation rate.
After a 72 hour wasp exposure, larvae from each plate were dissected and scored for the presence of an encapsulated wasp egg or live wasp larva, to assay the encapsulation rate.
Data analysis and statistics.
To analyze the effect of knockdown of proteases on wasp egg encapsulation rate, we used generalized linear models with quasibinomial errors to test for an effect of genotype, and then we performed Dunnett’s post hoc tests to compare each of the experimental genotypes to the control genotype. All statistics were done in the R statistical computing environment (R Core Team, 2020) using the “multcomp” (Hothorn
et al.
, 2008), “plyr” package (Wickham, 2011). Graphs were produced using the “ggplot2” package (Wickham, 2009).