王泽焕、卢欣、路群山、张佩2012/06/08

• 哺乳动物肠道大约有 100万亿个细菌，它们在肠道代谢过程中扮演着很重要的角色。然而，如此庞大数量的细菌在与肠道维持共生关系中是如何避免潜在的有害免疫应答反应的呢？

• 本文，我们发现了一种分泌型抗菌凝集素 RegIIIγ，它在维持着微生物菌群与肠道表皮间的空间分布中发挥着重要作用。同时，我们发现与野生型小鼠相比， RegIIIγ缺陷型小鼠不存在细菌与肠道表皮之间地理隔离，同时还伴随着表皮细菌的富集增长，以及宿主对细菌的免疫应答反应增强。

• 以上结果表明， RegIIIγ 通过调节细菌与肠道之间的空间分布，来维持宿主与细菌之间的共生关系。

Abstract

Introduction

Complex communities of intestinal bacteria inhabit the mammalian intestine, m

aking essential contributions to host metabolism.It remains unclear how these large

and diverse bacterial populations are maintained at homeostasis without negative

health consequences such as chronic immune activation andin flammation

Much of our current understanding of intestinal homeostasis is built around the idea tha

t the microbiota continuously interact with the intestinal immune system and that microb

iota species composition dictates the balance between proinflammatory and tolerogenic

immune responses

However, studies of the spatial relationships between microbiota and host point to t

he existence of a more general strategy for maintaining homeostasis.

Nevertheless, little is known about the immune mechanisms that promote host-bacterial segregation and m aintain homeostatic spatial relationships between hostand microbiota.

To explore the role of innate immunity in maintaining host-microbiota segregation

以上的结果表明依赖MyD88的先天性免疫效应因子限制了小肠黏膜微生物的数目，促进了小肠内部微生物与宿主之间明显的空间隔离。

Myd88

Identify the MyD88-dependent mechanisms that promote host-microbial segregation

Myd 88fl/fl Mice — harboring a floxed Myd88 allele

MyD88Δ IEC Mice = Villin - Cre Mice + Myd 88fl/fl Mice

Identify the MyD88-dependent mechanisms that promote host-microbial segregation

MyD88IEC mice lost the clear segregation no statistically significant differences in luminal bacterial loads

Thus, epithelial cell Myd88 is required to limit bacterial colonization of the mucosal surface in the small intestine but does not regulate overall numbers of colonizing bacteria

结果表明，在小肠粘膜表面限制细菌的繁殖生长中，上皮细胞Myd88起着必不可少的作用，但也不能够调节控制所有的菌群的生成

To determine whether epithelial cell Myd88 was also sufficient to maintain physical separation of host and microbiota

Vil- Myd 88Tg — transgenic mice that expressed Myd88 under the control of the IEC-specific villin promoter, then background whit Myd88−/−

to produce mice with IEC-restricted Myd88 expression

small intestinal tissues were probed with an anti - FLAG antibody

To determine whether epithelial cell Myd88 was also sufficient to maintain physical separation of host and microbiota

Vil-Myd 88Tg mice exhibited restoration of the distinct zone no significant differences

in overall numbers of luminal bacteriafewer mucosa-associated bacteria

Overall, our findings show that epithelial cell Myd88 is both necessary and sufficient to limit bacterial colonization of the mucosal surface

Establishing that the maintenance of homeostatic host-bacterial spatial relationships is an epithelial cell–intrinsic function

A plausible mechanism by which intestinal epithelial cells could limit bacterial-mucosal contact is through the production of secreted antibacterial proteins

MyD88 controls the expression of several keyantimicrobial proteins in Paneth cells

We hypothesized that RegIIIγ might be one epithelial cell– derived MyD88dependent factor that limits bacterial colonization of the mucosal surface

RegIIIγ

Whether RegIIIγ expression correlates with the physical segregation of microbiota and host

？

Whether RegIIIγ expression correlates with the physical segregation of microbiota and host

Myd88IEC mice showed reduced expression of RegIIIγ mRNA and protein relative to Myd88fl/fl littermates

Vil-Myd88Tg mice showed increased RegIIIg expression relative t o Myd88−/−littermates

Epithelial cell–intrinsic MyD88 signaling controls RegIIIγ expression, and this expression correlates with the physical separation of the microbiota from

the host mucosal surface

What is the role of RegIIIγ in maintaining host-microbial homeostasis ?

To directly assess the role of RegIIIγ in maintaining

host-microbial homeostasis

在 RegIIIγ−/− 鼠中， RegIIIγ 不能进行转录和蛋白表达。

Q-PCR analysis of RegIII transcripts in terminal ileum of wild-type and RegIIIγ−/−mice.

RegIIIγ was detected in distal small intestine using a polyclonal antibody raised against a peptide unique to RegIIIγ.

Defective in production of RegIIIγ perturbed spatial relationships between

the microbiota and the host mucosal surface in RegIIIγ−/− mice.

FISH analysis of wild-type and RegIIIγ−/−mice using a universal bacterial 16S rRNA gene probe(green).

免疫荧光分析结果表明 , RegIIIγ 的缺失会干扰微生物与宿主黏膜表面的空间关系，表现为与野生型相比粘膜相关细菌的数目显著增加。

Quantification of total ileal mucosa–associated bacteria by Q-PCR determination of 16S rRNA gene copy number.

Quantification of total ileal luminal bacteria by Q-PCR determination of 16S rRNA gene copy number.

In contrast, we did not detect significant differences in

luminal bacterial numbers .

luminal bacteria were quantified by Q-PCR determination of 16S rRNA gene copy number in the terminal ileum.

RegIII 的活性仅局限于黏膜表面微环境，而对肠腔细菌数目的影响很小。

There no defects in microbiota localization in the colons of RegIIIγ−/− mice.

在 RegIIIγ−/− 鼠结肠中没有发现微生物的空间位置没有变化，进一步研究发现，在结肠中 RegIIIγ 的表达量与小肠相比明显减少。

Thus, RegIIIγ is a key MyD88-dependent mechanism that limits bacterial colonization of the small intestinal mucosal surface and

maintains homeostatic spatial relationships between microbiota and host

According to the previous study ， the antibacterial activity of RegIII is restricted to Gram-positive bacteria in vitro.

So we predicted that RegIII−/− mice would exhibit a preferential increase in the abundance of mucosa-associated Gram-positive bacteria.

We therefore compared the abundance of specific bacterial groups in RegIIIγ−/− mice and wild-type littermates.

Q-PCR quantification of specific bacterial groups.Bacteria were recovered from the ileal mucosal surface.

Comparison of cohoused wild-type and RegIIIγ−/− mice by FISH using an SFB-specific probe (red).

RegIIIγ 在 体 内 只 对 G+ 菌 有 抑 菌 活 性。

And then ,we compare the luminal bacterial communities by Q-PCR and compare the phylum by pyrosequencing of 16S rRNA genes.

Quantification of total ileal luminal bacteria by Q-PCR determination of 16S rRNA gene copy number.

与野生型鼠相比， RegIIIγ−/− 鼠肠腔中主要 G+ 和 G- 菌群的丰度没有明显的区别，且微生物的种类也基本相似。

Mean relative abundances of bacterial phyla from the luminal communities of RegIIIγ-/-and wild-type mice.

Wild-type RegIIIγ−/−

These results establish that RegIIIγ limits associations between Gram-positive members of the microbiota and the small intestinal epithelial surface.

Further, they reveal that RegIIIγ activity is restricted to the mucosal surface niche and has little impact on luminal bacteria

We next asked whether RegIIIγ−/− mice exhibit enhanced activation of adaptive immune responses as a consequence of the increased numbers of mucosa-

associated bacteria

IgA is a critical adaptive immune effector that is secreted into the intestinal lumen

Whether RegIIIγ−/− mice exhibit enhanced activation of adaptive immune responses

RegIIIγ−/−小鼠 IgA免疫应答反应的提高随着抗菌素的加入而消失。

Whether RegIIIγ−/− mice exhibit enhanced activation of adaptive immune responses

结果表明， RegIIIγ−/−小鼠 TH1免疫应答反应的提高随着抗菌素的加入而消失。通过以上结果我们可以得出结论， RegIIIγ并不能直接影响宿主免疫应答反应，

而是通过改变肠道微生物的空间分布而达到的。