All living organisms are constantly exposed to various microbes, such as virus and bacteria, and vertebrates have developed sophisticated defense system against them, i.e. innate immunity and acquired immunity. Innate immunity is the first line of defense against infectious pathogens, mediated by antigen presenting cells (APCs), such as macrophages and dendritic cells (DCs). Acquired immunity is the late response process mediated by highly diverse antigen receptors on T cells or B cells, and antibodies produced by B cells. The diversity of antigen receptors and antibodies is generated by gene rearrangements and allows responses to a wide variety of potential antigens. Innate immune responses play key roles in the regulation of acquired immune responses by producing a battery of cytokines.

Innate immune responses are initiated by APCs that recognize specific structure of a invading pathogens by patter recognition receptors, Toll-like receptors (TLRs), which recognize a molecular pattern of pathogens, e.g. nucleic acids or components of bacterial cell walls. Binding of such pathogens to TLRs leads to activation of the transcription factors, such as nuclear factor kappa B (NF-KB) and interferon regulator factors (IRFs). They induce the production of pro-inflammatory cytokines and chemokines, which activate innate and adaptive immune responses against invading pathogens. Therefore, it is important to understand the regulatory mechanisms of cytokine expressions from APCs via TLRs.

NF-KB plays a central role in regulating the induction of pro-inflammatory cytokines by TLRs. NF-KB is composed of homo- or hetero-dimer of NF-x13/Rel family proteins, including p65 and p50. The activity of NF-KB is strictly regulated by a member of inhibitor of NF-KB (IKB) proteins. All IKB proteins are characterized by their multiple ankyrin repeat domains and IxBa, is a prototypical member of this family. In unstimulated cells, IxBa forms a complex with NF-KB and inhibits the nuclear translocation of NF-KB, keeping it as an inert complex in the cytoplasm. Stimulation of TLRs induces phosphorylation and ubiquitin-dependent degradation of IxBa, leading to release of NF-x13. Free NF-KB then translates into the nucleus and initiatestranscription of target genes, including pro-inflammatory cytokines and chemokines, by binding to -KB sites in their promoters or enhancers.

In addition to such canonical IKB proteins that are present in the cytoplasm and inhibit the NF-xB activity, a distinct class of IKB proteins has been recently found. Those atypical IKB proteins, including BCL-3, IKBNS and IKB1, are generally not expressed in unstimulated cells. As they are expressed in the nucleus by TLR stimulation and regulate the NF-KB-mediated transcriptional activity, they are also called as nuclear IKB proteins. Studies on those nuclear IxBs, especially using knockout mice, clearly show that these molecules play important roles in the expression of pro-inflammatory cytokines in the innate immune responses.

In our laboratory, microarray analysis of bone marrow derived DCs (BMDCs) was performed to findnovel molecules involved in the regulation of inflammation. Among many genes expressed in BMDCs, I found one novel gene encoding ankyrin-repeats. This gene had been registered as Ankrd42 in databases, but its function had not been described. Since ankyrin-repeats were known to be important for the interaction with NF-KB, I decided to characterize this protein in detail. The protein encoded by the gene consists of 516 amino acid residues and has 8 ankyrin repeats in the NH2-terminal region. As it was structurally similar to IKB proteins, I named it IKB1 and further analyzed its expression and functions.

IKBri mRNA was rather ubiquitously expressed in all the tissues examined and highly expressed in the brain, lung, testis and ovary, and expressed not only in DCs but also macrophages, T cells and B cells. As described above, IKB proteins are categorized into two groups, cytoplasmic IKBs and nuclear IKBs. Through the immunofluorescence staining and biochemical fractionation, I found that IKBy was predominantly expressed in the nucleus, indicating that IKBy is a nuclear IKB protein. In contrast to the previously characterized nuclear IKB proteins that are highly inducible by LPS in macrophages, IKari was only slightly induced by stimuli and rather constitutively expressed in macrophage cell line Raw264.7 cells. These results indicate that IKBy is a unique nuclear IKB that is constitutively and ubiquitously expressed in various tissues and blood cells.

One key feature of IKB proteins is their interaction with NF-KB components, and nuclear IKB proteins are known to interact with p50 rather than p65. Co-immunopreciptation assay revealed that kiln was co-immunoprecipitated with p50 subunit of NF-KB, but not with p65. This result indicates that IKIIri associates with a p50 homodimer or a heterodimer of p50 with an NF-KB subunit. Since it is known that nuclear IKB proteins prefer to interact with the p50 subunit of NF-KB, rather than p65, these results suggest that IKBy is characteristically similar to the nuclear IKB proteins, rather than typical cytoplasmic IKBs. I also analyzed the interaction between IkByand DNA by using avidin-biotion-conjugated DNA-binding (ABCD) assay. IKB11 interacted with the IL-6 promoter and also interacted with NF-xB subunits in response to LPS, suggesting that IkBy forms a transcriptional complex on the IL-6 promote to act astranscriptional cofactor.

To address the transcriptional activity of IkBy, I employed luciferase reporter gene assay using the NF-x13 binding site and found that IkBy enhanced the NF-KB-mediated transcription, suggesting that IKBri positively regulates the NF-KB's activity. To reveal the physiological role of I kBy as a regulator of NF-KB, I knocked down the expression of IkBy in Raw264.7 cells by using siRNA. Knockdown of IkBy suppressed the production of various pro-inflammatory cytokine mRNA, including TNFa, IL-6, IL-113, G-CSF, GM-CSF, and CXCL-2, indicating that IkBy regulates the NF-KB's transcriptional activity. It is known that there are at least two types of NF-KB-mediated gene expression in response to TLR signaling, i.e. primary and secondary response genes. Primary response genes, such as TNFa and IL-1B, are expressed immediately in response to TLR stimulation in the absence of protein synthesis, while the expression of secondary response genes, such as IL-6, G-CSF and GM-CSF, requires newly synthesized mediators of NF-KB and occurs after the primary responses. Knockdown experiments indicate that IKBri regulates both primary and secondary response genes. Because IkBy is constitutively expressed in the nucleus, it may regulate the primary response genes without protein synthesis.

In contrast to IkBy, IkBc, another nuclear IKB, is known to be rapidly induced by TLR signaling without protein synthesis, and newly synthesized IkBc, is essential for the induction of a subset of TLR-dependent secondary response genes. Since knockdown of 'Kari showed little or no effects on the expression of IkBNS and IkB, IkBy may affect secondary response genes by a mechanism independent of such nuclear IkB proteins, and both IkBy and IkB seem to be required for the expression of secondary response genes. In conclusion, IkBy is a new nuclear IkB protein that contributes to NF-KB-mediated transcription as a nuclear cofactor, and plays an important regulatory role in innate immune responses by regulating the expression of inflammatory genes. Although this study has focused on the innate immune responses of macrophages to TLR signaling, ubiquitous expression of IkBy suggests that it may also play an important role for regulation of NF-KB signaling in non-immune cells.

本論文は5つのChapterからなる。Chapter 1はIntroductionであり免疫系の概説および本論文で解析したNF-kBシグナル、核IkBについてこれまでの知見が述べられている。Chapter 2でのMaterials and methodsに続き、Chapter 3のResultsにて本研究にて明らかにしたIkByの機能解析の結果が、Chapter 4のDiscussionでは結果をふまえた上で、NF-kBシグナルにおけるIkByの意義および既存のIkBとの差異について考察が記されており、最後にChapter 5にてConclusionが述べられている。

申請者の所属する研究室では抗原提示細胞群に着目し、免疫系におけるサイトカイン産生およびサイトカインシグナル伝達機構などの免疫制御機構の解析を進めている。申請者は抗原提示細胞などに発現する新規のIkBファミリー分子であるIkByを見いだし、Toll-like receptor (TLR)下流での炎症性サイトカイン産生において中心的な役割を果たすNF-kBの転写活性制御機構との関わりについて、詳細に解析している。

本論文では、自然免疫における炎症性サイトカイン産生の制御機構を明らかにすることを目的として、TLRの下流におけるシグナル伝達機構について解析している。自然免疫を司るマクロファージや樹状細胞には種々のTLRが発現しており、これらの受容体による病原体成分の認識により、様々な炎症性サイトカインの発現が誘導される。TLRの下流で機能する主要なシグナル分子であるNF-kBは、炎症応答に関わる数多くの遺伝子の発現制御に深く関わっており、NF-kB活性制御の分子機構を理解することは自然免疫応答を理解する上で非常に重要である。NF-kBはいくつかのIkBファミリー分子によってその活性が正又は負に制御されていることが知られており、特に核内においては核IkBと呼ばれる分子郡によってその転写活性が制御されている。申請者は、マイクロアレイ法を用いた網羅的遺伝子発現解析の結果、新規のIkB分子を見いだしている。この分子は特徴的なankyrin repeatを持ち既存のIkBファミリー分子と構造的に類似するが、機能などについては不明であったため、この分子をIkByと命名しさらに解析を進めた。IkByは、既知のIkBファミリー分子と異なり、TLR刺激による発現誘導を受けず恒常的にマクロファージに発現していた。さらに、IkByは核に局在することから、核IkB分子として機能する可能性が考えられた。そこでさらなる生化学的、分子生物学的な解析を行った結果、IkByはNF-kBの転写活性を正に制御することで、様々な炎症性サイトカインの発現を促進することを明らかにしている。NF-kBにより発現が制御される遺伝子群は、その発現パターンから少なくとも二つのグループ、すなわちTNFy代表される刺激に対して非常に早い応答を示す初期応答遺伝子群と、IL-6のように刺激に対して徐々に発現が誘導される二次応答遺伝子群に分類される。既知の核IkBは初期および二次応答遺伝子いずれかの発現制御に関わるが、IkByはいずれの遺伝子応答にも関与することを明らかにしている。またTLRの下流では、MyD88、およびTRIFを介した二つのシグナル経路によりNF-kBを活性化するが、様々なTLR刺激に対するIkByの機能を解析した結果、IkByはTRIFを介したシグナル経路にてNF-kBの転写活性を正に制御することが明らかにしている。本研究は、新規の核IkB分子としてIkByを同定し、それがNF-kBの転写活性を正に制御してサイトカイン産生を調節するという新たな免疫制御機構の存在を明らかにしており、自然免疫研究の進展に寄与することが大いに期待される。

なお、本論文Chapter 3は伊藤寛明、宮島篤との共同研究であるが、申請者が主体となって実験および考察を行った物であり、申請者の寄与が十分であると判断する。よって、博士(理学)の学位を授与できると認める。