The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus

Stephanie M. Robert, Benjamin C. Reeves, Emre Kiziltug, Phan Q. Duy, Jason K. Karimy,

M. Shahid Mansuri, Arnaud Marlier, Garrett Allington, Ana B.W. Greenberg, Tyrone DeSpenza, Jr., Amrita K. Singh, Xue Zeng, Kedous Y. Mekbib, Adam J. Kundishora, Carol Nelson-Williams, Le Thi Hao, Jinwei Zhang, TuKiet T. Lam, Rashaun Wilson, William E. Butler, Michael L. Diluna, Philip Feinberg, Dorothy P. Schafer, Kiavash Movahedi, Allen Tannenbaum, Sunil Koundal, Xinan Chen, Helene Benveniste, David D. Limbrick, Jr., Steven J. Schiff, Bob S. Carter, Murat Gunel, J. Marc Simard, Richard P. Lifton, Seth L. Alper, Eric Delpire, and Kristopher T. Kahle

Cell press, February 16, 2023

http://doi.org/10.1016/j.cell.2023.01.017

Abstract:

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF ‘‘cytokine storm’’, elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

한글 초록 요약본:

 뇌실막총 (Choroid plexus, ChP)은 뇌척수액 (CSF)을 생성하고 면역 감지 기능을 수행하는 주요 기관임. 본 연구에서는 감염성 및 출혈성 수두증 (Post-infectious hydrocephalus, PIH/ Post-hemorrhagic hydrocephalus, PHH)이 공통적으로 ChP에서 Toll-like receptor 4 (TLR4)를 중심으로 하는 면역 반응과 과도한 CSF 생성으로 이어진다는 점을 밝힘. 이 과정은 면역세포에서 분비된 사이토카인이 ChP 상피세포의 SPAK-NKCC1 수송체계를 활성화하면서 발생하며, 결과적으로 수두증으로 유발함. 유전자 조작이나 면역조절제를 통해 이러한 경로를 차단하면 수두증의 발생을 예방할 수 있음. 본 연구는 수두증을 면역 조절 기반의 치료로 접근할 수 있는 가능성을 제시함

한글 논문 요약본 (Intro, Methods, Results)

1) Introduction: ChP는 뇌실 내 CSF를 생성하고, 혈액-CSF 장벽으로서 면역세포의 유입을 조절함. 그러나 ChP의 면역-분비 기능이 수두증 병태생리에 어떻게 기여하는지는 명확하지 않음. 기존에는 PIH와 PHH가 주로 CSF의 재흡수 감소, 유출 경로 폐색 등으로 인한 결과로 여겨졌지만, 최근 연구에서는 CSF 생성 증가도 중요한 원인일 수 있음이 제기됨. PIH는 보통 LPS를 가진 박테리아 (E.coli 등)에 의한 감염으로, PHH는 혈액 분해 산물 (heme)에 의한 염증 반응으로 유발됨. 이들은 모두 TLR4 수용체를 통해 면역 반응을 유도하며, 비슷한 사이토카인 프로파일과 면역세포 반응을 보임. 본 연구는 PIH 및 PHH 모델을 구축하고, ChP의 면역-분비 반응을 다중 오믹스 (multi-omics) 분석을 통해 규명함.

2) Methods

 2-1) 모델 구성: Wistar 랫드를 이용해 E. coli 또는 LPS를 뇌실에 직접 주입하여 PIH 모델을, 자가 혈액을 뇌실에 주입하여 PHH 모델을 구축

 2-2) CSF 생산 측정: 뇌실 내 유출 경로를 차단한 뒤 실시간으로 CSF 생산량을 측정함.

 2-3) 분석 기법: bulk RNA-seq, 단일세포 RNA-seq (scRNA-seq), proteomics, 면역조직화학염색, FACS 분석을 병행함.

 2-4) 면역조절 실험: SPAK 유전자 결손, TLR4 KO 쥐 및 mtor 억제제 rapamycin을 이용해 치료 효과를 평가함.

3) Results

3-1) CSF 과다 생성: LPS나 E. coli (+LPS)를 주입한 PIH 모델에서 CSF 생성이 2.5배 증가하였으며, 이는 NKCC1 억제제인 bumetanide에 의해 억제됨.

3-2) TLR4 의존적 반응: TLR4 결손 쥐에서는 CSF 생성 증가와 ChP 염증 반응이 모두 억제되었음.

3-3) 면역 반응 활성화: ChP에서 Iba1+, CD68+ 대식세포가 급격히 증가하였고, 이는 사이토카인 폭풍 (cytokine storm) 유발과 연관됨. scRNA-seq에서는 주변에서 유입된 CD45+, CD3+ T세포 및 혈액 유래 대식세포가 ChP에 축적됨이 확인됨.

3-4) SPAK-NKCC1 경로 활성화: ChP 상피세포에서 TNF-α, IL-1β 등의 사이토카인 수용체가 발현되며, SPAK가 NKCC1을 활성화해 CSF 생성을 증가시킴. SPAK이 NKCC1 외에도 Na+/K+ ATPase, KCNJ13등 다수의 이온수송체 복합체와 결합함.

3-5) PHH와의 병리학적 유사성: PIH와 PHH 모두 ChP 면역세포 침윤, SPAK 활성화, pnkcc1 증가, CSF 생성 증가, 그리고 염증성 사이토카인 유사한 프로파일을 공유함.

3-6) 치료 가능성: mtor 억제제 rapamycine은 사이토카인 발현, 면역세포 침윤, CSF 생성 및 pspak/ pnkcc1 발현을 모두 억제함. PIH/PHH 발병 전후에 투여해도 효과있음.

Figure 1. Models of E. coli post-infectious hydrocephalus exhibit ChP-mediated CSF hypersecretion