Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster

Abstract

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. Here, we characterize the Drosophila tuSz¹ mutant strain, which mounts an aberrant immune response against its own fat body. We demonstrate that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Our data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals.

Keywords: autoimmunity; innate immunity; protein N-glycosylation; self-recognition; self-tolerance.

Fig. 1.

The tuSz self-encapsulation phenotype. (A–C) In tuSz¹ mutants at 28 °C, posterior fat body tissues are melanized during the third-instar larval stage (A), and the melanization persists through the pupal (B) and adult stages (C). The self-encapsulation phenotype is morphologically similar to the encapsulation of a Leptopilina clavipes (parasitoid wasp) egg by a w¹¹¹⁸ control larva (D). Brightfield (E), eGFP (F), and mCherry (G) images of fat bodies dissected from tuSz¹, eater-eGFP, msn-mCherry larvae demonstrate that plasmatocytes (marked by eGFP expression) and lamellocytes (marked by mCherry expression) interact with fat body tissue that is undergoing melanization. Brightfield (H), GFP (I), and merged (J) images of fat bodies dissected from tuSz¹; LanA-GFP larvae show that self-encapsulated fat body tissue nevertheless maintains intact ECM (marked by GFP expression).

Fig. 2.

Phenotypic characterization of tuSz flies. (A) Penetrance of the self-encapsulation phenotype of w¹¹¹⁸ (control), tuSz¹, and tuSz¹/w¹¹¹⁸ heterozygous flies raised at the indicated temperature. *P < 0.05 compared to w¹¹¹⁸ at 28 °C. (B–E) Plasmatocyte number (B), lamellocyte number (C), podocyte number (D), and crystal cell number (E) of the indicated genotypes at the indicated temperatures. *P < 0.05 compared to w¹¹¹⁸ at each temperature. P values for A and C were determined by Dunnett’s test. P values for B, D, and E were determined by Welch’s two-sample t test.

Fig. 3.

The role of JAK-STAT signaling in tuSz¹ mutants. (A) An amino acid sequence alignment within the JH2 domain of Hop and the Hop orthologs GA14036 from Drosophila pseudoobscura and JAK2 from humans shows the missense D. melanogaster Hop^Sz allele. (B–E) Brightfield (B) and GFP (C) images of immune cells from Stat92E-GFP larvae raised at 28 °C show no JAK-STAT pathway activity. Brightfield (D) and GFP (E) images of immune cells from tuSz¹; Stat92E-GFP larvae raised at 28 °C show JAK-STAT pathway activity. (F) Penetrance of the self-encapsulation phenotype in tuSz¹ and tuSz¹; Stat92E⁰⁶³⁴⁶/+ flies raised at 28 °C. *P < 0.05 compared to tuSz¹. (G) Penetrance of the self-encapsulation phenotype in tuSz¹; msn-GAL4; UAS-GAL4^RNAi and tuSz¹; msn-GAL4; UAS-Stat92E^RNAi flies raised at 28 °C. *P < 0.05 compared to tuSz¹; msn-GAL4; UAS-GAL4^RNAi. P values were determined by generalized linear models. (H and I) Lamellocytes (marked by mCherry expression) aggregate around large self-encapsulations in tuSz¹, msn-mCherry larvae (arrow in H) but are dispersed throughout hop^Tum, msn-mCherry larvae (I). Tissue self-encapsulations are not seen in hop^Tum, msn-mCherry larvae, but small melanized nodules are common (arrow in I).

Fig. 4.

Mapping the hypothesized tuSz¹ self-tolerance mutation. (A) Schematic demonstrating the mapping of the tuSz¹ recessive mutation on the X chromosome. Lines are listed by strain name. The hop gene locus is indicated on the chromosome. (Top) Interrupted segments illustrate the location of genome deletions in the indicated Drosophila strains. Black lines indicate deletions that fail to complement the tuSz¹ self-encapsulation phenotype. Gray lines indicate complementing deletions. (Middle) Blocks represent duplicated regions of the X chromosome. Black blocks indicate duplications that rescue the tuSz¹ self-encapsulation phenotype, and gray blocks indicate failure to rescue. (Bottom) The deletion and duplication lines indicate a small locus for the tuSz¹ SAMP mutation. Candidate genes are shown. (B) Penetrance of the self-encapsulation phenotype of tuSz¹ flies in combination with the indicated duplication raised at 28 °C. *P < 0.05 for the indicated comparisons. (C) Penetrance of the self-encapsulation phenotype of tuSz¹ heterozygous flies in combination with w¹¹¹⁸ controls or the indicated GCS1 allele raised at 28 °C. *P < 0.05 for the indicated comparisons. P values for B and C were determined by Dunnett’s test.

Fig. 5.

Fat body GCS1 expression and protein N-glycosylation. (A–D) Posterior fat body tissue dissected from early third-instar w¹¹¹⁸ (A and B) and tuSz¹ (C and D) larvae raised at 28 °C prior to tissue melanization. (A and C) Brightfield images of dissected fat body tissue demonstrating the absence of tissue melanization. (B and D) Corresponding fluorescent images of α-GCS1 antibody staining. (E–J) Magnified posterior fat body tissue dissected from early third-instar larvae and stained by FITC-WGA to assay protein N-glycosylation. w¹¹¹⁸ fat body from larvae raised at 28 °C (E) or 18 °C (F) is positive for FITC-WGA staining. tuSz¹ fat body tissue is negative for FITC-WGA at 28 °C (G) but positive at 18 °C (H). tuSz¹/w¹¹¹⁸ heterozygous (I) and hop^Tum (J) larvae raised at 28 °C are positive for FITC-WGA staining, indicating that hop mutations do not affect fat body ECM protein N-glycosylation.

Fig. 6.

Loss of GCS1 leads to loss of self-tolerance. (A and B) Posterior fat body tissue dissected from larvae raised at 25 °C and stained with FITC-WGA to assay protein N-glycosylation. Control c833-GAL4; UAS-GAL4^RNAi tissue is positive for FITC-WGA staining (A). c833-GAL4; UAS-GCS1^RNAi shows a mosaic loss of FITC-WGA staining, where * marks fat body cells with decreased staining (B). (C) tuSz¹; c833-GAL4; UAS-GCS1^RNAi flies raised at 25 °C show melanized self-encapsulations (indicated by the arrow). (D) Penetrance of the self-encapsulation phenotype in control tuSz¹; c833-GAL4; UAS-GAL4^RNAi and tuSz¹; c833-GAL4; UAS-GCS1^RNAi flies raised at 25 °C. *P < 0.05 compared to tuSz¹; c833-GAL4; UAS-GAL4^RNAi. (E) Penetrance of the self-encapsulation phenotype in hop^Tum/+, hop^Tum/+; Mgat1^KG02444/+, and hop^Tum/+; α-Man-IIb^MI09613/+ flies raised at 28 °C. *P < 0.05 compared to hop^Tum/+. (F) hop^Tum/+; Mgat1^KG02444/+ flies raised at 28 °C show melanized self-encapsulations (indicated by the arrow). (G) hop^Tum/+; α-Man-IIb^MI09613/+ flies raised at 28 °C show melanized self-encapsulations (indicated by the arrow). P values (D and E) were determined by generalized linear models.

Fig. 7.

Models describing the necessity for two independent signals in fly encapsulation responses. (A) Homeostasis is maintained in naïve wild-type larvae. (B) In tuSz¹ mutant larvae, immune cells are inappropriately activated by JAK-STAT pathway activation due to the hop^Sz gain-of-function mutation. The loss of protein N-glycosylation in posterior fat body tissue due to the GCS1^Sz mutation leads to loss of self-tolerance and tissue encapsulation. (C) In the model of self-tolerance described by Kim and Choe (55), the coupled phenotypes of loss of cell integrity and loss of ECM integrity are sufficient to disrupt self-tolerance. (D) Immune cells are activated following parasitoid wasp infection, presumably due to the wound-mediated activation of JAK-STAT signaling. SAMP-presenting host tissues are protected from encapsulation, and wasp eggs may be targeted for encapsulation because they are missing the ECM N-glycosylation SAMP.