Beruflich Dokumente
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Correspondence
christoph.scheiermann@med.
uni-muenchen.de
In Brief
Lymphocyte trafficking through lymph
nodes and lymph is an important immune
surveillance mechanism of the body.
Druzd et al. (2017) demonstrate that this
trafficking occurs in a circadian manner
and that adaptive immune responses are
also time-of-day dependent and are
ablated when circadian clock function is
lost in T cells.
Highlights
d Lymphocyte numbers in lymph nodes and lymph oscillate
over the course of the day
Article
*Correspondence: christoph.scheiermann@med.uni-muenchen.de
http://dx.doi.org/10.1016/j.immuni.2016.12.011
120 Immunity 46, 120132, January 17, 2017 2017 The Author(s). Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A B
CD4+ T cell
1.5
****
CD8+ T cell **** 4
8 CD4+ T cell
****
1.5
LN count (x106)
LN count (x106)
3 6
LN count (%)
1.0 1.0
2 4
50
0.5
**
0.5
1 2
LN Blood CD8+ T cell DD
**** B cell **** DL
0 0 0.0
**** LD
0.0 0
ZT 1 5 9 13 17 21 1 ZT 1 5 9 13 17 21 1 ZT 1 5 9 13 17 21 1 CT 1 5 9 13 17 21 1
Mes
C ** Ing **** D E
6 Axi
** Sup Com
** 6 Egress block 1.2 2.5
combined counts (x106)
CD4+ T cell
12 Homing block
CD8+ T cell
LN count (x106)
Control
LN count (x106)
LN count (x106)
Lymphocyte Homing Is Dependent on Oscillations in Lymphatic Egress from Lymph Nodes Is under Circadian
Lymphocytes and Microenvironment Control
We next used adoptive transfer techniques to determine whether Because our data indicated that, in addition to a rhythmic homing
lymphocyte homing to the LN was occurring in a rhythmic component, lymphocyte egress might counterbalance LN oscilla-
manner. LN infiltration of lymphocyte subpopulations peaked tions (Figures 1D and 1E), we quantified lymphocyte numbers in
around night onset and remained low during the day (Figure 2A). lymph fluid by cannulating efferent lymphatic vessels. Prominent
To define whether oscillations were determined by lymphocyte- rhythms in cellular counts were detected, peaking at ZT9 and
intrinsic and/or microenvironmental signals, we adoptively exhibiting a low at ZT21 (Figure 4A). These oscillations were
transferred cells harvested at ZT5 (day) or ZT13 (night) into observed for different lymphocyte populations (Figure 4A and Fig-
LD-entrained recipients at either ZT5 or ZT13. While day (cells) ures S2H and S2I) and were bona fide circadian in nature as they
into day (recipient) transfers exhibited the lowest homing ca- persisted in constant darkness (Figure 4B). Rhythms were not due
pacity and night into night transfers the highest, a mixed to higher lymph volume or flow rates at different times of the day
contribution of both lymphocyte and microenvironment timing (Figure S2J). To verify whether oscillations in lymph cellularity
was observed in the day into night and night into day were truly attributable to rhythmic egress and not secondary to
chimeras (Figure 2B). A screen for oscillations of promigratory rhythmic input into LNs (Figure 2A), we transferred lymphocytes
factors on T and B cells revealed that expression of the chemo- at different times of the day, blocked subsequent LN entry and
kine receptor CCR7 exhibited rhythmicity peaking at ZT13 quantified their transit through the LN into lymph over time (Mandl
(Figure 2C) while the adhesion molecules CXCR4, CD11a, and et al., 2012). A higher LN retention capacity of cells injected at ZT13
L-selectin showed either no oscillations or not for all lymphocyte was observed compared to ZT5 and a less rapid accumulation of
subpopulations (Figures S2A and S2B). In addition, expression cells in lymph (Figures 4C and 4D and Figure S3), demonstrating
analyses of whole lymph node mRNA and extracellular protein lymphocyte egress to be highly rhythmic. This effect resulted in
on HEVs revealed oscillatory amounts of the chemokine longer LN half-lives of cells injected at ZT13 (CD4: 12 hr, CD8:
CCL21, a ligand for CCR7but not CXCL12 (not shown)being 12 hr, B cells: 16 hr) compared to ZT5 (CD4: 9 hr, CD8: 9 hr, B cells:
high around night onset (Figures 2D and 2E). HEVs also exhibited 13.5 hr) (Figures 4C and 4D and Figure S3). T- and B cell-specific
rhythmic expression of ICAM-1 but not of PNAd (Figures S2C BMAL1-deletion ablated oscillations in lymph, indicating the
and S2D). Oscillations in lymphocyte chemokine receptors importance of cell-autonomous clocks also for lymphocyte egress
were critical for rhythmic homing because a titrated, short pre- (Figure 4E and data not shown). Of importance, adoptively trans-
treatment of adoptively transferred cells with pertussis toxin ferred BMAL1-deficient CD4+ T cells exhibited no time-of-day var-
(PTX) (Lo et al., 2005), an inhibitor of chemokine receptor iations in their LN half-life (Figure 4F), demonstrating the relevance
signaling, ablated rhythmicity (Figure 2F). To investigate the of T cell clocks in their rhythmic trafficking behavior.
involvement of CCR7 in this process, we analyzed total lymph Using a mathematical approach, we assessed whether oscil-
node cellularity of CCR7-deficient mice, as well as the rhythmic latory LN counts could be modeled with only either homing or
homing capacity of isolated CCR7-deficient cells. Ccr7 / mice egress to be rhythmic or whether both components needed to
exhibited no oscillations in lymph node cell counts while also ex- oscillate. Although oscillations were also observed when only
hibiting the expected lower overall numbers (Forster et al., 1999) one component was rhythmic, the best fit was achieved when
(Figure 2G). In addition, Ccr7 / cells failed to show rhythmic both homing and egress were assumed to oscillate, thus sup-
lymph node homing (Figure 2H). These data demonstrated that porting our experimental data (Figure 4G and Figure S4). In sum-
lymphocyte recruitment to LNs is determined by rhythms in leu- mary, lymphocyte clocks and the time-of-day entry of cells into
kocytes and the microenvironment, along with in-phase expres- LNs have functional consequences for LN transit and egress
sion of the CCR7-CCL21 receptor-ligand axis. into lymph.
Circadian Clocks Control Cellular Oscillations in Rhythmic Lymphocyte Egress Depends on Oscillatory
Lymph Nodes S1pr1 Expression
LNs exhibit oscillations of clock genes (Figure 3A), prompting S1P-receptors are critical in regulating lymph node egress
us to investigate the role of lymphocyte clocks in their migra- (Cyster and Schwab, 2012). We therefore investigated whether
D E
F G H
Figure 2. Rhythmic Lymphocyte Homing Is Dependent on Oscillations in Both Lymphocytes and Microenvironment
(A) Lymph node homing of lymphocyte populations over the course of the day, normalized to peak times; n = 317 mice, one-way ANOVA.
(B) Adoptive transfer of lymphocyte populations using donor and recipient mice kept at ZT5 or ZT13; n = 617 mice, one-way ANOVA with Tukeys multiple
comparisons test.
(C) Oscillations of CCR7 surface expression on lymphocyte subpopulations in LN; n = 35 mice, one-way ANOVA.
(D) Q-PCR analysis of LN Ccl21 amounts over 24 hr; n = 35 mice, one-way ANOVA.
(E) Quantification and images of expression of CCL21 on HEV over 24 hr in constant darkness (CT, circadian time: the corresponding light and dark phase are
indicated); n = 318 mice, one-way ANOVA. Scale bar represents 50 mm.
(F) LN homing of lymphocytes harvested at ZT5 or ZT13 and treated with or without pertussis toxin (PTX); n = 511 mice, one-way ANOVA with Tukeys multiple
comparisons test.
(G) Lack of oscillations in LN cellularity of Ccr7 / mice; n = 4 mice, unpaired Students t test.
(H) Lack of rhythmic LN homing of Ccr7 / cells into WT hosts; n = 56 mice, unpaired Students t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data are
represented as mean SEM. See also Figure S2.
expression of S1P-receptor family members exhibited oscilla- ure 5A and Figure S5A), which coincided with high lymphocyte
tions using quantitative PCR (Q-PCR). All S1P receptors exhibited egress. In addition, FTY720, as well as the S1P1-specific func-
robust diurnal oscillations peaking between ZT1 and ZT9 (Fig- tional antagonist SEW2871 strongly down-modulated lymphatic
1.0 1.0
0.5
0.5 0.5
* * *
0.0 0.0 0.0
ZT 1 5 9 13 17 21 1 ZT 1 5 9 13 17 21 1 ZT 1 5 9 13 17 21 1
4 2.0 3 2.5
* ZT19
* ZT19
**** ns
2.0 ****
ns
3 1.5
LN count (x106)
2
1.5
2 1.0
1.0
1
1 0.5
0.5
0 0.0 0 0.0
ZT 1 7 13 19 1 ZT 1 7 13 19 1 Ctrl Bmal1-/- Ctrl Bmal1-/-
2.0
*
1.0 1.2
Homed cells (x104)
ns 1.5 1.0
1.5 ns
0.8
1.0
1.0 0.5 0.6
0.4 Ctrl
0.5 0.5
CD4+ T cell Bmal1-/-
*
0.2 Bmal1-/- *
CD8+ T cell Bmal1-/-
0.0 0.0 0.0 0.0
Donor (ZT) 5 13 5 13 5 13 5 13 ZT 1 7 13 19 ZT 1 7 13 19
Ctrl Bmal1-/- Ctrl Bmal1-/-
4 50 * 50
1
2 25
DD
0 0 LD 0
0
ZT 1 5 9 13 17 21 1 ZT 1 5 9 13 17 21 1 CT 1 5 9 13 17 21 1 0 6 12 18 24
Hr post block
D Lymph E CD4+ T cell CD8+ T cell F G
4 2.0 100 ZT5
ZT5 ** ZT13 5
Lymph count (x103/l)
Cells remaining (%)
100 ZT13
LN count (x106)
ns
ns
75 * 60 3
2 1.0
50 40 2
** 1 0.5
25 20 1
0 0 0.0 0 0
0 6 12 18 24 ZT 1 7 13 19 ZT 1 7 13 19 Ctrl Bmal1-/- ZT 0 12 24
Hr post block Bmal1-/- Bmal1-/-
egress in a time- and concentration-dependent manner (Figures the proper timing of lymphocyte egress (Figures 5F and 5G
5B and 5C and Figure S5B). The observation that FTY720-treated and Figure S5C). Importantly, no diurnal oscillations were
animals exhibited reduced but still rhythmic lymph cellularity indi- observed in amounts of S1P in efferent lymph (Figure 5H) or in
cated a daytime-sensitive role for S1P1 in mediating lymphocyte S1P synthesizing or degrading enzymes in lymph node (Fig-
exit. Rhythmic expression of S1pr1 was ablated in BMAL1-defi- ure S5D), suggesting that oscillatory expression of the receptor
cient CD4+ T cells, pointing toward a regulation of the gene by (S1P1) and not its ligand (S1P) was the driver for rhythmic
the circadian clock (Figure 5D). To investigate this in more detail, lymphocyte egress. Together, these data demonstrate a critical
we performed an in vitro assay, in which the promoter region of role for S1P1 in mediating circadian lymphocyte egress from
S1pr1 was cloned in front of the luciferase (luc) reporter gene. lymph nodes into efferent lymph.
Luciferase activity in HEK293 cells transfected with the S1pr1-
luc reporter was decreased after co-transfection of increasing Relevance of Circadian Oscillations in Lymph Node
amounts of Bmal1 and Clock expression plasmids (Figure 5E). Cellularity
This demonstrated that expression of S1pr1 is regulated by We hypothesized that oscillatory lymphocyte counts in LNs
BMAL1 and CLOCK. might have functional consequences in a potential time-of-day
To confirm the role of S1P1 in the time-of-day-dependent dependence of adaptive immune responses. We therefore
egress genetically, we generated T cell-specific mice that tested whether the activation status of lymphocytes in LNs var-
were heterozygous for S1pr1 in order not to completely ied over the course of the day. More activated T cells were pre-
block lymphocyte egress (Matloubian et al., 2004) but to titrate sent in LNs at night onset as assessed by CD69 staining, coin-
S1P1 amounts, as loss of one allele had been demonstrated to ciding with higher overall lymphocyte counts at this time
result in haploinsufficiency (Lo et al., 2005). S1pr1+/floxxCd4-cre (Figure 6A). Because dendritic cells (DCs) are key antigen-pre-
mice exhibited no more oscillations in LN counts and altered senting cells critical in the activation of lymphocytes and the gen-
lymph rhythmicity, demonstrating the importance of S1P1 in eration of adaptive immune responses (Girard et al., 2012), we
D E F
G H
next investigated whether these cells also exhibited oscillatory LN counts in the autoimmunity model of EAE. Mice immunized
LN counts. Migratory DC cellularity showed strong oscillations during the late light phase (ZT8, when cell counts are high in
peaking in phase with lymphocytes (Figure 6B and Figure S6A). LNs, Figures 1A and 1C) showed a dramatically accelerated
These data point to the existence of a concerted circadian disease progression 2 weeks later, with higher clinical scores
migration pattern of antigen-bearing (DCs) and antigen-recog- compared to late night-immunized animals (ZT20, when LN
nizing (T cells) cells in LNs. counts trough) (Figure 6C). Differences in disease scores
Recent evidence indicates that the immune system can were associated with higher immune cell infiltration and demy-
respond to challenges in a rhythmic fashion (Fortier et al., elination in the spinal cord at the peak of the disease (Figures
2011; Gibbs et al., 2014; Nguyen et al., 2013; Scheiermann 6D and 6E). We detected elevated interleukin-2 (Il2) mRNA
et al., 2012; Silver et al., 2012b). We therefore investigated amounts (Figure 6F) and a higher number of IL-17 producing
the pathophysiological significance of circadian oscillatory as well as very-late antigen (VLA)-4 integrin positive CD4+
E F G H
I J K
L M
(D) Quantification of demyelination in lumbar spinal cord sections; n = 5 mice, unpaired Students t test.
(E) Luxol Fast Blue staining of lumbar spinal cord sections of mice immunized at ZT8 (top) or ZT20 (bottom) at the peak of the disease showing demyelinated areas
(arrows), scale bar represents 200 mm (left images), 100 mm (right images).
(F) LN IL-2 mRNA after EAE induction; n = 35 mice, unpaired Students t test.
(G) LN counts of IL-17+ and VLA-4+ CD4+ T cells after EAE induction; n = 5 mice, unpaired Students t test.
(HJ) Diurnal profiles of inguinal lymph node counts of total CD4+ T cells (H), naive CD4+ T cells (I), and activated CD4+ T cells (J) on day 2 after EAE induction; n = 4
mice, two-way ANOVA with Bonferronis post hoc test.
(K) EAE disease scores and EC50 values in T cell-specific Bmal1 / mice immunized at ZT8 or ZT20; n = 5 mice.
(L) Inguinal lymph node counts of CD4+ T cells, and CD8+ T cells in T cell-specific Bmal1 / mice at ZT15 on day 2 after EAE induction; n = 45 mice, unpaired
Students t test.
(M) Schematic diagram of circadian lymphocyte migration through lymph nodes. At night onset, increased homing due to higher CCR7 amounts leads to
enhanced lymphocyte counts in the lymph node. During the day, higher S1pr1 expression induces the egression of lymphocytes into efferent lymph. *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001. All data are represented as mean SEM. See also Figure S6 and Movie S1.