Spotlight
The Intestinal Mucus
Layer Comes of Age
Shipra Vaishnava1,*
The mucus layer is critical in limit-
ing contact between host and the
complex bacterial consortia that
colonize the intestine. A recent
paper in Cell Host and Microbe
provides comprehensive insight
into the dynamics of mucus layer
maturation upon bacterial coloni-
zation of germ-free (GF) mice that
have implications for studies on
host-microbe interaction involving
colonization of GF mice.
Several factors limit the ability of gut bac-
teria to access host tissues. Top amongst
them is the mucus layer, which is com-
posed of mucin glycoproteins that are
constantly secreted by specialized epithe-
lial cells called goblet cells into the gut
lumen. Until recently the mucus layer
was thought to be a physical barrier of
constant nature and without much dyna-
mism. Contradictory to this view, Johans-
son et al. have revealed a highly transient
nature of mucus layer upon colonization of
GF mice [1]. This study highlights yet
another nuanced way by which interac-
tions with commensal bacteria are nego-
tiated at the host-microbe interface.
Adhesion of gut bacteria to the intestinal
epithelium is critical for induction of inflam- following colonization of GF mice with
matory immune responses. Recently, the
induction of Th17 cells was shown to
require adhesion of segmented filamen-
tous bacteria (SFB) to epithelial cells
[2,3]. Several mechanisms are in place
to minimize bacterial interactions with
the host mucosal surfaces. In the small
intestine, spatial segregation between
bacteria and intestinal tissues is main-
tained by secretion of specialized antimi-
crobial proteins such as RegIIIg by The mucus of the GF small intestine, as
intestinal epithelial cells. Mice lacking compared to colonized small intestine, is
RegIIIg are unable to keep bacteria from
accessing mucosal surfaces and as result
show increased adaptive immune
responses [4]. In the large intestine, spatial
compartmentalization of bacteria is main-
tained by two structurally distinct layers of
mucus [5]: The inner mucus layer closest
to the epithelial cells layer delineates a
bacteria-free protected zone, whereas
the outer mucus layer contains large num-
bers of bacteria. Mice genetically engi-
neered to lack the mucin glycoprotein
Muc2 do not have this bacteria-free zone
and suffer from spontaneous intestinal
inflammation, emphasizing the impor-
tance of the mucus barrier in maintaining
a symbiotic relationship with the intestinal
microbiota [6]. Although the role of the
mucus barrier in protecting against bacte-
rial invasion by pathogenic bacteria has
long been acknowledged, its role in regu-
lating interactions with intestinal micro-
biota have come to light only recently.
Studies show that the mucus layer serves
as distinct microbial niche for bacteria that
can digest complex glycoproteins to use
as carbon source. In this way, mucus layer
secretion can be considered a mechanism
by which host can select for bacteria that it
associates with [7].
The composition of gut bacteria that asso-
ciate with the mucus layer can have huge
impact on the permeability of the mucus
barrier; however, how gut microbiota
impact the organization of the mucus layer
was not well understood. By performing a
comprehensive analysis of the physico-
chemical properties of the mucus layer
murine cecal contents, Johansson et al.
show that it takes a surprisingly long time
to obtain relative homeostatic levels of
major mucins and their spatial expression.
Interestingly, throughout this maturation
event the mucus layer shows dynamic
and transient shifts in physical properties,
chemical makeup and bacterial commu-
nities that associate with it.
anchored to the goblet cells and requires
enzymatic cleavage for release [8].
Johansson et al. show that it takes 4–5
weeks after colonization for the mucus to
show this detached phenotype. In the
large intestine, where the mucus layer
organizes into two layers, it takes 6 weeks
for the inner mucus layer to become
impenetrable to bacteria-sized beads
(Figure 1). These results strongly suggest
that signals from colonizing microbes
greatly alter mucin production and glyco-
sylation over the period of 8 weeks, and
that it takes approximately 6 weeks after
colonization for the mucus barrier of GF
mice to become impenetrable to bacteria.
The results further demonstrate that the
changes in properties of the mucus barrier
are closely associated with shifts in bac-
terial composition during the colonization
period: a transient shift in Firmicutes and
Bacteroidetes was observed during the
colonization period, with Bacteroidetes
peaking at week 4 after colonization.
The epithelial-adherent SFB were shown
to colonize only during early time points,
and contracted after 5 weeks of coloniza-
tion. These microbial fluctuations were
associated with heightened levels of anti-
microbial proteins such RegIIIb and RegIIIg
and secretion of IgA. Furthermore, the
data suggest that the maturation of the
mucus barrier involves some robust and
long-lasting mechanisms, as its perme-
ability remained unaffected even after 3
weeks of treatment with four antibiotics.
Overall, this study points towards a com-
plex, well-programmed, and intricate sys-
tem by which host and microbe regulate
the mucus layer in the gut.
Similar bacterial colonization of sterile
mucosal surfaces takes place immediately
following birth in humans. In infants, a high
variability is observed within the gut micro-
biota during the first year of life, which
begins to stabilize at 2 years of age [9].
However, little is known about the effect of
this massive bacterial colonization event
on the development of the mucus layer in
the gastrointestinal tract of infants. It is
possible that initial colonization events
Trends in Immunology, January 2016, Vol. 37, No. 1 3
(A) Early (2–3 weeks) (B) Late (6–8 weeks) towards the design and interpretation
of experiments aimed at understanding
the interactions between the intestinal
Mucus layer: Aached, permeable Mucus layer: Detached, impermeable
microbiota and the immune system. Fur-
Firmicutes Bacteroidetes Firmicutes Bacteroidetes ther work on how bacteria regulate func-
tional properties of the mucus barrier is
certain to open doors to new avenues of
research in the area of host-microbiome
interactions.

1
Molecular Microbiology and Immunology Department,
Brown University, Providence, RI, USA

Immune Anmicrobial proteins *Correspondence: shipra_vaishnava@brown.edu

Immune IL1 beta (S. Vaishnava).
response IgJ, Pigr, C3, Tff3 response IL 17 http://dx.doi.org/10.1016/j.it.2015.12.002

Figure 1. Mucus layer properties during early and late stages of colonization of GF mice. A. First 2–3
weeks post colonization secreted mucus layer is attached to epithelial cells and is permeable to bacteria sized
beads. There is no bacteria free zone close to the epithelium. Major colonizers of mucus layer during this period
are firmicutes specially SFB. The immune responses at this time are characterized by increased AMP production
and enhanced IgA secretory mechanisms. B. 6–8 weeks post colonization the mucus layer attains the detached
configuration similar to conventional mice. The mucus layer becomes impermeable to bacteria sized beads and
has enhanced bacteria free zone near the epithelial cells. The bacteria colonizing the mucus layer at this time are
mainly Bacteroidetes and there is a switch in immune response to interleukin (IL) 1b and IL-17 driven.
are critical for establishing mucus proper- diversity of mucus residing bacterial com-
ties, which in turn have long-lasting effects munities is needed to understand how the
on host-microbe homeostasis. By employ- bacterial composition alters mucus prop-
ing gnotobiotic mouse models, we can erties. This understanding could have a
References
1. Johansson, M.E. et al. (2015) Normalization of host intestinal
mucus layers requires long-term microbial colonization. Cell
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microbes to intestinal epithelial cells. Cell 163, 367–380
3. Sano, T. et al. (2015) An IL-23R/IL-22 circuit regulates epi-
thelial serum amyloid A to promote local effector Th17
responses. Cell 163, 381–393
4. Vaishnava, S. et al. (2011) The antibacterial lectin RegIII-
gamma promotes the spatial segregation of microbiota
and host in the intestine. Science 334, 255–258
5. Johansson, M.E. et al. (2011) The two mucus layers of colon
are organized by the MUC2 mucin, whereas the outer layer is
a legislator of host-microbial interactions. Proc. Natl. Acad.
Sci. U.S.A. 108 (Suppl 1), 4659–4665
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neously develop colitis, indicating that MUC2 is critical for
colonic protection. Gastroenterology 131, 117–129
7. Li, H. et al. (2015) The outer mucus layer hosts a distinct
intestinal microbial niche. Nat. Commun. 6, p8292

begin to understand the interplay between
specific components of gut microbiota and
the physicochemical properties of the
intestinal mucus layer. Moreover, a detailed
understanding phylogenetic and functional
profound and translatable impact on ther-
apies for inflammatory diseases by guid-
ing development pre- and pro-biotic
interventions to reinforce the mucus bar-
rier. These findings also have implications
8. Schutte, A. et al. (2014) Microbial-induced meprin beta
cleavage in MUC2 mucin and a functional CFTR channel
are required to release anchored small intestinal mucus.
Proc. Natl. Acad. Sci. U.S.A. 111, 12396–12401
9. Backhed, F. et al. (2015) Dynamics and stabilization of the
human gut microbiome during the first year of life. Cell Host
Microbe 17, 690–703

4 Trends in Immunology, January 2016, Vol. 37, No. 1