[Background and introduction]

Inflammatory response represents a critical aspect of in vivo defense system, which can be triggered in response to threat with both intrinsic and extrinsic origin. Upon microbial challenge, an inflammatory response is essential in eliminating invading pathogens and wound repair. However, unresolved, chronic inflammation could be detrimental to the host, therefore should be tightly regulated to avoid further damage to the tissues involved. Recently, accumulating evidence has suggested that inflammation could also be induced by a handful of host factors in the absence of microbial signals, causing pathological symptoms as seen in stroke, atherosclerosis, diabetes and so on. It is of great importance to understand the nature of the endogenous immunogen and the molecular basis for such sterile inflammatory response.

In this study, Drosophila, with its vast collection of genetic tools and analyzing techniques available, is utilized to decipher this phenomenon in vivo. The relatively simple composition of body tissues makes them well fitted for studies at organismal level, whereas it is difficult to investigate the inter-tissue communication in vivo in mammalian system. More importantly, although devoid of the highly specific adaptive immunity that governs a great part of the mammalian immune system, Drosophila possesses an effective and conserved innate immune system that actually holds the key to multiple aspects in defense response, thus has become an attractive tool in elucidating the fundamental principles in immunity.

Apoptosis, a well-conserved mechanism of cell suicide, is considered immuno-silent and plays fundamental roles in physiological processes such as morphogenesis, homeostasis as well as host defense. In Drosophila, Dronc, the only initiator caspase, is the key regulator for most apoptosis; mutants for dronc exhibit severe defects and die at mid-pupal stage. In the present study, I conducted a detailed characterization of sterile inflammation on the basis of genetic manipulation in Drosophila dronc null mutants and demonstrated that canonical Toll signaling is responsible to mount a potent response to endogenous immunogenic factors released upon apoptosis deficiency, and postulate that Drosophila caspase mutants would serve as a model to enable further elucidation of the mechanism of sterile inflammation in vivo.

[Methods and Results]

1. Sterile inflammation manifested as systemic activation of Toll pathway in dronc(ΔA8) mutants

Previously a deletion line for dronc was generated by P element mobilization in my laboratory. The homozygous mutants are refractory to apoptotic stimuli and show complete lethality during pupal development. With this dronc(ΔA8) mutant line, I then sought to explore whether inflammatory response ensues due to the apoptosis deficiency. In third instar larvae, quantitative RT-PCR results revealed a profound induction of the Toll signaling, one of the prominent immune regulators induced by fungi and gram-positive bacteria, with significantly elevated production of Drosomycin (Drs, an anti-fungal peptide frequently used as a hallmark of Toll pathway activation in Drosophila), and to a lesser extent, Metchnikowin (Mtk, an anti-microbial peptide) (Figure 1A). On the contrary, Imd pathway, the other Drosophila immune signaling activated mainly by gram-negative bacteria, remains unaffected. Likewise, no discernable induction could be observed for the other stress pathways tested. Moreover, in dronc(ΔA8) mutants, the concomitant elevation of the signaling molecules in Toll pathway demonstrates a robust and systemic activation of this pathway (Figure 1B). Given the important role of Toll signaling in combating invasive pathogen, I attempted to determine whether microbial infection plays a part by looking at the immune activation in germ free animals (Figure 1C). The qRT!PCR results showed that removing the bacteria load didn't abolish Toll activity, indicating in dronc(ΔA8) mutants, it is the host factors, rather than bacterial elicitors, that trigger the immune activation.

2. Apoptotic deficiency causes constitutive NF-!B activation.

In metazoan, caspases are produced as inactive precursors and become activated by upstream caspase through proteolytic cleavage, which depends on an active cysteine site. I then assessed whether the catalytic activity of Dronc is crucial in maintaining immune-homeostasis.

Drosophila fat body, the functional equivalent of mammalian liver, is the central tissue for systemic production of AMPs, which are transcriptionally regulated by corresponding NF-!B/Rel factors. Accordingly, in mutant fat body, I could detect substantial amount of nuclear localized Dorsal, the Rel protein downstream of Toll pathway, suggesting a constitutive activation of Toll intracellular cascade (Figure 2A-B). Furthermore, Dorsal immuno-staining demonstrates that oversxpression of wild type dronc could readily reverse Dorsal nuclear import in mutant background (compare figure 2C-D), whereas a loss of function allele (dronc C→A, Figure 2E) with an alanine substitution at the active cysteine site couldn't. These results suggest that catalytic activity is crucial to suppress the Toll signaling activation.

3. In dronc(ΔA8) mutant, Toll pathway activation is mediated by active form of the cytokine-like factor Spatzle.

In infectious activation of Toll, extracellular recognition of stimuli leads to the processing of the endogenous cytokine Spatzle to its active form that comprises C-terminal 106 amino acids, which could bind Toll receptor with high affinity and relay the signal intracellularly via recruiting the adaptor protein dMyd88 (Figure 1B). I then explored whether, in dronc(ΔA8) mutants, host factor-directed NF-κB activation is mediated through such canonical cascade. By genetically combining the mutation for either spz or dMyd88 with dronc(ΔA8) background, I showed that both factors are essential in mediating Toll signaling, for the induction of Drs was completely abolished in individuals doubly mutated for dronc and immune factors (Figure 3A).

As genetics indicates spz is necessary to mediate Toll signaling, I next tried to determine whether the proteolytic processing occurs in the mutant hemolymph. To this end, I overexpressed tagged Spatzle protein (Spz-HA) using a hemocytes driver (pxn-Gal4) and assessed its cleavage in hemolymph by western blotting (Figure 3B). The active Spz was detected as a 19kDa protein in mutant hemolymph. Taken together, these results suggest that impairment in caspase leads to the processing of the Drosophila cytokine Spatzle, causing constitutive activation of the canonical Toll signaling.

4. Inactivation of Toll signaling rescues hemocyte defects.

In addition to humoral immunity that is characterized by AMP secretion from the fat body, the cellular immunity represents another vital force in Drosophila host defense system. In third instar larvae, two distinct fractions of hemocytes could be observed: circulating hemocytes that reside in the hemolymph, and the sessile hemocytes that attach to the inner surface of the integument. In an effort to reveal whether loss of dronc could influence cellular immunity, I observed a significant elevation in number of circulating blood cells in mutant larvae, which could be partially attributed to Toll activation (Figure 4A). In addition, the mutants exhibit a profound disruption of the banded pattern for sessile hemocytes, as visualized by GFP-expressing blood cells through their translucent skin (Figure 4B). Taken together, the above results implicate caspase function in the regulation of hematopoietic tissue, likely through Toll signaling.

[Summary and discussion]

In addition to microbial determinants, there is increasing evidence suggesting that host factors would activate immune system as a result of environmental or physiological stressor. In this study, I characterized a sterile inflammatory response with respect to both humoral and cellular immunity using Drosophila mutants for the initiator caspase, Dronc. The results suggest that caspase activity might be required to maintain homeostasis in terms of suppressing chronic immune signaling. In dronc(ΔA8) larvae, Toll innate immune signaling is specifically induced following the generation of the active form of the cytokine Spatzle. This phenomenon shows profound analogy to the induction of TLR signaling by altered or abnormal host factors in mammalian system. The fundamental role of TLR/IL-1 signaling in endogenous factor-induced host response is starting to be understood, and Drosophila dronc(ΔA8) mutants provide us with a genetic accessible tool that enables elucidation of the molecular basis of sterile inflammation in vivo.

In vertebrate system, in addition to an induced production of pro-inflammatory cytokines and chemokines from the effected site, sterile inflammation is also marked by the extensive activation and recruitment of immune cells such as neutrophils and macrophages. Drosophila hemocytes constitute the major body of mobile immune cells, and are indispensible for the cellular arm of defense response. In a manner similar to mammalian immuno-surveillance cells, Drosophila hemocytes play significant parts during host defense response and maintenance of homeostasis under normal state. In dronc(ΔA8) mutant, the profound changes of behavior associated with these insect blood cells exemplified a simultaneous disturbance of the cellular immunity by sterile factors originated from the host.

The physiological interaction between apoptotic cell death and inflammatory response, two of the most important biological processes in organisms, underlies pathological principles of human diseases such as cancer growth and massive degeneration, thus has been under intensive investigations. In vertebrate models, multiple stimuli are shown to converge on caspase function to promote inflammatory responses. Overall, the results of present study representing a non cell-autonomous role of apoptotic caspase in suppressing inflammation might highlight a novel connection between cell death control and inflammation.

Figure 1 dronc(ΔA8) mutants display hyper-activated Toll signaling in the absence of bacterial infection.(A) Relative induction of stress responsive genes was analyzed by quantitative RT-PCR. (B) Toll signaling in Drosophila. Arrows indicate the enriched signaling proteins in mutants. (C) Germ free animals was verified by LB plating and the activation of immune pathways was assessed using qRT-PCR. *p<0.05,***p<0.001

Figure 2 Immuno-staining of fat body reveals intracellular localization of the Rel protein Dorsal for Toll pathway. Genetic background: a: w1118, b: dronc(ΔA8), c: Act-Gal4/+; dronc(ΔA8), d: Act>drone(WT); drone(ΔA8), e: Act>dronc(C>A); dronc(ΔA8), Scale bar; 20μm

Figure 3 In dronc(ΔA8) mutants, transduction of Toll signal requires both the cytokine-like ligand Spatzle and the cytosolic adaptor dMyd88. (A) Mutation in either spz or dMyd88 abolished Drs induction under dronc(ΔA8) genetic background. Between different mark: p<0.01, between identical mark: p>0.05, (B) Western blotting detects active form of the Spz in dronc(ΔA8) hemolymph. Z: full-length zymogen, C: cleaved form.

Figure 4 The impairment of cellular immunity. (A) Circulating hemocyte counts from indicated genotypes indicates a drastic hyperplasia in dronc(ΔA8) mutant. (B) Disruption of the banded pattern for sessile hemocytes. blood cells associated with the integument are visualized using pxn-Galr4, UAS-GFP. Band organization is indicated by '*'; arrows point to the hematopoietic organ-lymph gland.

プログラム細胞死であるアポトーシスは自己の組織のターンオーバーやダメージを受けた細胞を除去するのに必要である。その実行はカスパーゼ(caspase)をはじめとした一群のアポトーシス遺伝子によって厳密かつ多重に制御されており、生体の恒常性維持において重要な役割を果たしている。一方、その破綻はネクローシス(necrosis)や組織傷害を引き起こし、内在性ストレスの原因になる可能性が示唆されている。しかし、危険因子を含む内在性因子を介した生体ストレス応答の組織、個体レベルでの解析は未だ少なく、自己由来の物質に応答するシグナルメカニズムの多くは明らかになっていない。本研究では、遺伝学的な解析手法が高度に発達したショウジョウバエを用いて、アポトーシス不全系統であるイニシエーターカスパーゼdronc変異体において内在性因子による自然免疫応答の活性化を見いだした。

ショウジョウバエ胚でのアポトーシスは、イニシエーターカスパーゼdroncを経由して、細胞死実行カスパーゼが活性化されておこる。まず、野生型およびdronc null変異体dronc(ΔA8)3齢幼虫に対して、定量的RT-PCRを行い、ストレス反応経路の転写産物のmRNAの量を測定することによって、ストレス経路の活性化を検討した。この解析から、dronc(ΔA8)変異体においては、主要な自然免疫機構であるToll経路が特異的に活性化していることを見いだした。病原性シグナルによるToll経路の活性化は種を超えて広く保存されており、自然免疫システムの中で中心的な役割を果たしている。ショウジョウバエにおいて、Toll経路は細胞膜に存在するToll様受容体を介して活性化され、真菌や大部分のグラム陽性細菌の感染に対する体液性の防御応答に必要であり、その下流で抗菌ペプチドを産生する。Dronc(ΔA8)変異体におけるToll経路の活性化はGerm-free(無菌)ショウジョウバエでもみられることから、この現象が細菌感染とは無関係であり、自己由来の内在性因子によって引き起こされることが示唆された。また、dronc(ΔA8)変異体の脂肪体(ショウジョウバエの主要免疫組織)における免疫染色の観察から、Toll経路の下流にある転写因子Dorsal(NF-κB)の核移行が起きていることが明らかになった。さらに、変異体に野生型droncを強制発現させた個体において、Dorsalの核移行は阻害されたが、droncの活性中心に変異を持つ変異体を発現した個体では見られなかった。以上の結果から、dronc(ΔA8)変異体におけるToll経路の活性化が、カスパーゼ酵素活性不全によるものであることが確認された。さらに、変異体脂肪体におけるToll経路活性化がdronc欠損による細胞自律的な反応ではないことが脂肪体におけるモザイククローン解析によって分かった。ショウジョウバエの3齢幼虫ではさまざまな組織のアポトーシスが知られ、その不全による組織傷害やストレス因子の体液中への放出が考えられた。

感染によるショウジョウバエToll経路の活性化はToll受容体を介して引き起こされる。このプロセスにおいては、Toll受容体と細胞外リガンドSpatzleの切断による活性化が必要である。dronc(ΔA8)変異体の体液では細胞外リガンドSpatzleが切断されていた。さらに遺伝学的解析の結果では、Spatzle及びToll受容体細胞内領域に結合するアダプター分子dMyD88の変異によって、Toll経路の活性化が起きなくなることが明らかになった。これら結果から、アポトーシス不全により炎症性液性因子が体液中に放出され、それによって古典的なToll経路が活性化していることが示唆された。

ショウジョウバエの自然免疫応答は、抗菌ペプチドの産生による体液性免疫および血球細胞による細胞性免疫の2つに大きくわけられる。dronc(ΔA8)変異体において、細胞性免疫について検証したところ、循環血球細胞数の増加が観察された。さらに、血球細胞においてもToll経路の活性化が起きていることが、Dorsalの免疫染色によって示された。dronc(ΔA8)変異体におけるToll経路の活性化が血球細胞の増殖あるいはリンパ腺からの放出に関わっていると考えられる。

アポトーシスは生体恒常性の維持に重要であり、その破綻は組織傷害を引き起こし、内在性ストレスの発生及び自己免疫反応に関与することが示唆されている。自己組織からの内在性因子による自然免疫の活性化は、糖尿病や肥満症、アテローム性動脈硬化などの幾つかの疾患に見られている。しかし自己由来の免疫原の実体、認識機構及びシグナル伝達のメカニズムの多くは未だ不明である。ショウジョウバエは獲得免疫を持たないため、ヒトとよく保存されている自然免疫のみで多様な刺激から生体を防御している。本研究で見出したアポトーシス変異体における内在性因子による自然免疫系の活性化は、この問題に遺伝学的にアプローチできる優れたモデルであり、今後分子レベルでのメカニズム解明が期待される。以上より、本研究は博士(薬学)の学位に値すると判定した。