Lymphoid tissue inducer – like cells are
T h e J o u r n a l o f E x p e r i m e n t a l M e d i c i n e
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35
BRIEF DEFINITIVE REPORT
N aive T cells undergo diff erentiation on signals received from the TCR and cytokine receptors and diff erentiate to specialized subsets character-ized by their production of signature cytokines. Th17 cells produce IL-17 or IL-17A and IL-17F, which are major mediators of infl ammation and are critical for host defense against extracellular bacteria and fungi ( 1, 2 ). IL-6, IL-21, and TGF- ? 1 are critical factors that promote Th17 cell dif-ferentiation ( 1, 3 ). Although IL-23 was originally thought to be important for inducing naive
CD4
+ T cells to become Th17 cells, naive CD4 + T cells do not express IL-23Rs ( 4 ). Rather, IL-23 is now thought to aff ect the expansion, main-tenance, and pathogenicity of Th17 cells ( 5 ). IL-23 also induces IL-17 production from ? ? T cells and invariant NKT (iNKT) cells ( 6, 7 ). Regardless of exactly how IL-23 works, current
evidence clearly argues that IL-23 – m ediated
IL-17 production is crucial in host defense and in the pathogenesis of autoimmune diseases ( 8 – 10 ).
I L-6, IL-21, and IL-23 share the ability to activate Stat3, which was shown to be critical for Th17 cell diff erentiation in mouse and man ( 3, 11, 12 ). Stat3 directly regulates the I l17 and I l21 genes but also regulates IL-23R expression ( 3, 12 ). Furthermore, the aforementioned cyto-kines acting via Stat3 induce the retinoic acid – r elated orphan receptor ? t (ROR ? t ), the master regulator of Th17 cell diff erentiation ( 13 ).
I n contrast to T cells, much less is known about the ability of innate cell subpopulations to produce IL-17. We report that splenic lym-phoid tissue inducer – l ike cells (LTi-like cells) constitutively express ROR ? t , IL-23R, CCR6, and aryl hydrocarbon receptor (AHR) ( 14 ), and produce IL-17 and IL-22. Interestingly,
CORRESPONDENCE
H iroaki Takatori: takatorih@https://www.sodocs.net/doc/2c10836149.html,
C .M. Tato ’ s present address is Dept. of Discovery Research, DNAX Research Inc., Palo Alto, CA 94304. G . Weiss ’ s present address is National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, M
D 20852.
L ymphoid tissue inducer – l ike cells are an innate source of IL-17 and IL-22
H iroaki T akatori , 1 Y uka K anno , 1 W endy T. W atford , 1 C ristina M. T ato , 1 G reta W eiss , 2 I vaylo I. I vanov , 3 D an R. L ittman , 3 and J ohn J. O ’ S hea 1
1 L ymphocyte Cell Biology Section, Molecular Immunology and Infl ammation Branch, National Institute of Arthritis
and Musculoskeletal and Skin Diseases, Bethesda, MD 20892
2 U niversity of Pennsylvania – N ational Institutes of Health Graduate Program, Bethesda, MD 20892
3 H oward Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University School of Medicine,
New York, NY 10016
T
he interleukin (IL) 17 family of cytokines has emerged to be critical for host defense as well as the pathogenesis of autoimmune and autoinfl ammatory disorders, and serves to link adaptive and innate responses. Recent studies have identifi ed a new subset of T cells that selectively produce IL-17 (Th17 cells; Bettelli, E., T. Korn, and V.K. Kuchroo. 2007.
C urr. Opin. Immunol. 19:652 – 657; Kolls, J.K., and A. Linden. 2004. I mmunity. 21:467 –
476), but the regulation of IL-17 production by innate immune cells is less well under-stood. We report that in vitro stimulation with IL-23 induced IL-17 production by
recombination activating gene (Rag) 2 ? / ? splenocytes but not Rag2 ? / ? common ? chain ? / ?
splenocytes. We found that a major source of IL-17 was CD4 + C D3 ? N K1.1 ? C D11b ? G r1 ?
C D 11c ? B 220 ? cells, a phenotype that corresponds to lymphoid tissue inducer – l ike cells (LTi-like cells), which constitutively expressed the IL-23 receptor, aryl hydrocarbon receptor, and CCR6. In vivo challenge with the yeast cell wall product zymosan rapidly induced IL-17 production in these cells. Genetic deletion of signal transducer and activator of tran-scription 3 reduced but did not abrogate IL-17 production in LTi-like cells. Thus, it appears that splenic LTi-like cells are a rapid source of IL-17 and IL-22, which might contribute to dynamic organization of secondary lymphoid organ structure or host defense.
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36
IL-17 AND IL-22 PRODUCTION BY SPLENIC LT I -LIKE CELLS | Takatori et al.
with IL-23 had no eff ect on IL-17A production; however,
IL-4 suppressed IL-23 – i nduced IL-17A production by WT
and Rag2 ? / ? splenocytes (Fig. S1 A, available at http://www
https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/jem.20072713/DC1). In addition
to memory CD4
+ T cells, iNKT cells are another important source of IL-17 ( 7 ), but such cells are lacking in Rag2
? / ? mice. Accordingly, anti-CD3/28 or ? -galactosylceramide ( ? -GalCer) stimulation induced IL-17A production by WT
splenocytes but not Rag2 ? / ? splenocytes (Fig. S1, B and C).
Collectively, these data suggest that although T and NKT cells are both major producers of IL-17, populations of non – T , non – B cells can also produce IL-17.
T o identify which population of cells might be responsible for IL-23 – d ependent IL-17 production by Rag2 ? / ?
spleno-cytes, we examined another genetic model, namely Rag2 ? / ? ? c ? / ? mice. In addition to lacking T and B cells, Rag2 ? / ? ? c ? / ?
mice also lack NK cells. We therefore cultured WT, Rag2 ? / ? ,
or Rag2
? / ? ? c ? / ? splenocytes in the presence of IL-23. Con-sistent with F ig. 1 A , IL-17A production was present in WT
splenocytes and reduced but not absent in Rag2 ? / ? splenocytes
( F ig. 1 B ). In contrast, IL-17A production by Rag2 ? / ? ? c ? / ?
splenocytes was essentially abrogated ( F ig. 1 B ). These results indicate that ? c -dependent, non – T , non – B cell populations in the spleen can produce IL-17 in vitro.
I L-23 has been shown to be induced by certain microbial products ( 15 ). Rag2 ? / ?
mice were therefore injected with zymosan to induce IL-23 production by DCs, which resulted in rapid IL-17A appearance in sera and spleens ( F ig. 1 C ). Thus, these data indicate that innate immune cells can gener-ate IL-17 in response to the fungal product in vivo.
I L-17 production by CD4 + C D3 ? C D11c ? B 220 ? LTi-like cells
T o delineate the population of IL-17 – p roducing cells, we next used intracellular cytokine staining. We used markers for a variety of innate immune cells expected to be present
in Rag2 ? / ? spleens but failed to detect IL-23 – m ediated
IL-17A production in granulocytes, macrophages, or DCs (Fig. S2, available at https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/jem .20072713/DC1). A prominent ? c -dependent lineage that rapidly produces cytokines upon stimulation is the NK cell.
Based on the response to IL-23 in Rag2 ? / ? splenocytes and
loss in Rag2 ? / ? ? c ? / ? splenocytes, we thought that NK cells
could be IL-17 producers. However, contrary to our expec-tations, the majority of IL-17A – p roducing CD3
? cells did not express NK1.1 ( F ig. 2 A ). Thus far, our results have indi-cated that neither T, NKT, NK cells (all ? c dependent) nor ? c -independent myeloid cells were a major source of
IL-17 production in Rag2 ? / ? spleens. We did observe that in
Rag2 ? / ? splenocytes stimulated with IL-23 ( F ig. 2 B , left), a considerable proportion of the IL-17A – p roducing non – T cells expressed CD4 ( F ig. 2 B , right).
C D4 + C D3 ? cells in the Rag2 ? / ? spleen comprise three
populations, including CD11C low B 220 + plasmacytoid DCs,
CD11C + B 220 ? conventional DCs, and CD4 + C D3 ? C D11c ? B220 ? cells (Fig. S3, available at https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/jem.20072713/DC1). As indicated in Fig. S2,
the yeast wall product zymosan elicited IL-17 production by LTi-like cells in vivo. Whether the production of IL-17 and IL-22 infl uences the architecture of secondary lymphoid or-gans (SLOs) and contributes to host defense will be important issues to examine in the future.
R ESULTS AND DISCUSSION
I L-23 induces IL-17 production by a population of common ? chain ( ? c ) – d ependent non – T , non – B cells
T o determine if there are considerable proportions of innate immune cells that produce IL-17, we examined Rag2
? / ? mice that have few T and B cells. We fi rst assessed whether
IL-17 production occurred in Rag2 ? / ? splenocytes after
stimulation with various cytokines. As shown in F ig. 1 A , IL-23 alone induced IL-17A production by WT splenocytes, presumably indicative of an eff ect on memory T cells. Con-sistent with the idea that memory T cells are lacking in
Rag2
? / ? mice, the eff ect of IL-23 was reduced in Rag2 ? / ? splenocytes ( F ig. 1 A ). However, Rag2 ? / ?
splenocytes still produced about one third as much IL-17A as WT splenocytes ( F ig. 1 A ). Other cytokines used separately or in combination
F igure 1. I L-23 and zymosan induce IL-17 production in a popu-lation of non – B , non – T cells. (A) WT or Rag2
?/? splenocytes were cul-tured in the presence of the indicated cytokines for 48 h, and IL-17A in culture supernatants was measured by ELISA. The data are means ± SD from duplicate cultures and are representative of four independent ex-periments ( n = 8). **, P < 0.01. (B) WT, Rag2
?/? , or Rag2 ?/? ?c ?/? spleno-cytes were cultured in the presence of IL-23 alone, and IL-17A in culture supernatants was measured by ELISA. The data are means ± SD from duplicate cultures and are representative of three independent experi-ments ( n = 6). *, P < 0.05; **, P < 0.01. (C) Sera and splenocytes were har-vested from Rag2 ?/? mice after injection of 5 mg zymosan or PBS
(Control). (left) IL-17A in sera was measured by ELISA. (right) The relative expression levels (/18S) of IL-17A mRNA were analyzed by quantitative PCR (q-PCR) and are depicted as fold induction relative to cells 1 h after treatment with zymosan. The data are means ± SD of results from two mice per group per time point and are representative of two independent experiments. ND, not detected.
JEM VOL. 206, January 19, 2009
37
BRIEF DEFINITIVE REPORT
to maintain cells for the initial 48 h, followed by IL-23 for an-other 6 h. As shown in Fig. S7 A, IL-17A and IL-17F mRNA
expression was potently induced by IL-23 in the presence of IL-7. These results suggest that LTi-like cells quickly enhance the production of IL-17 in response to IL-23, and this cyto-kine does not simply serve to keep these cells alive.
I t is well known that Stat3 is activated by IL-23 and other inducers of Th17 cell diff erentiation, and the absence of Stat3 abrogates IL-17 production by T cells ( 12 ). We therefore as-sessed the proportion of IL-17A – p roducing LTi-like cells in Stat3-defi cient mice. Not surprisingly, we found that IL-23 – m ediated IL-17A production was decreased in Stat3-defi cient
LTi-like cells of S tat3 fl /fl ; MMTV-Cre or S tat3 fl /fl ; CD4-Cre
splenocytes compared with those of S tat3 fl /fl splenocytes, al-though IL-17A production was not completely abrogated
( F ig. 3 B and not depicted). Because Cre-mediated deletion of STAT3 is > 90% in both systems (unpublished data), there seems to be both STAT3-dependent and -independent path-ways for the production of IL-17 in LTi-like cells. We also observed that IL-23 up-regulated mRNA expression of Stat3 and Stat4 but not GATA3 in LTi-like cells (Fig. S7 B). These results highlight the notion that the IL-17 production by LTi-like cells might be dependent on these factors.
L
Ti-like cells constitutively express IL-23R, AHR, and CCR6 T he complete receptor for IL-23 comprises the IL-12R ? 1 associated with the ligand-specifi c subunit IL-23R, and both subunits are required for the action of this cytokine ( 4 ). As
memory CD4 + T cells respond to IL-23 because of constitu-tive expression of its receptors ( 4 ), we evaluated mRNA
expression of IL-23R and IL-12R ? 1 in LTi-like cells. Consistent with their rapid responsiveness to IL-23, LTi-like cells constitutively expressed IL-23R mRNA at levels sig-nifi cantly greater than T cells ( F ig. 3 C ). LTi-like cells also expressed slightly higher levels of IL-12R ? 1 mRNA com-pared with those of memory CD4 + T cells ( F ig. 3 C ). In contrast, expression levels of IL-12R ? 2 mRNA in LTi-like
cells were low compared with those in memory CD4 + T
cells ( F ig. 3 C ). Also consistent with previous studies ( 16, 18 ), we noted higher expression levels of ROR ? t mRNA in LTi-like cells compared with T cells ( F ig. 3 C ). In contrast, expression of IL-6R ? and IL-17RA mRNA in LTi-like cells was much lower compared with that in T and B cells (unpublished data). Recently, the AHR has been reported to be expressed in Th17 cells ( 14 ). Interestingly, LTi-like cells also expressed AHR mRNA at levels equivalent to those in Th17 cells polarized for 72 h ( F ig. 3 C ). Furthermore, IL-23 up-regulated mRNA expression levels of IL-23R, ROR ? t , and AHR (Fig. S7 C).
C onsistent with previous studies ( 16, 18 ), splenic LTi-like
cells w ere f ound t o b e C D30L +
O X40L + ? c + I L-7R ? + T hy1.2 high C D44 high C D62L
low (Fig. S6). LTi-like cells are also known to express the chemokine receptors CXCR5 + and CCR7
+ ( 16, 18 ), and we again confi rmed expression of these in the splenic LTi-like cells (Fig. S6). Additionally, it has recently been shown that CCR6 identifi es a population of human
we did not detect IL-23 – m ediated IL-17A production by
CD11C + DC populations. The latter, CD4 + C D3 ? C D11c ? B220 ? cells, have been shown to be present in SLOs of fetal and adult mice and are termed LTi ’ s and LTi-like cells, re-spectively ( 16 ). Importantly, LTi-like cells are greatly reduced
in ? c ? / ? mice (
17 ). W e next determined whether isolated CD4 + C D3 ?
CD11c ? B 220 ? LTi-like subsets produced IL-17 ( F ig. 3 A , top). In fact, as shown in F ig. 3 A (bottom), we observed IL-17A production by isolated LTi-like cells in response to IL-23. Moreover, directly isolated LTi-like cells produced more IL-17A in the presence of PMA/ionomycin (Iono), which was dramatically enhanced by IL-23 ( F ig. 3 A , bottom). In contrast, LTi-negative subsets produced minimal IL-17A (Fig. S4, available at https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/
jem.20072713/DC1). Consistent with Fig. S2, isolated CD11c
+ cells did not produce IL-17A with IL-23 stimulation (unpub-lished data). Another source of LTi-like cells in an adult mouse is the intestinal lamina propria ( 16 ). Interestingly, LTi-like cells in the lamina propria from WT mice produced IL-17A after stimulation with PMA/Iono (Fig. S5). These results suggest that the ability to produce IL-17 is not unique to the splenic population.
O ne possible explanation for our observations is that IL-23 promoted in vitro survival of LTi-like cells rather than en-hancing IL-17 production by itself. LTi-like cells have been shown to express IL-7Rs (Fig. S6, available at http://www https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/jem.20072713/DC1) ( 16 ). There-fore, we modifi ed the cell-culture conditions by adding IL-7
F igure 2. C D4 + C D3 ? l ineage ? cells produce IL-17.Rag2 ?/? spleno-cytes were cultured in the absence (Control) or presence of IL-23 for 24 h.
The proportion of IL-17A – p roducing CD3
? cells was evaluated by intra-cellular staining. (A) NK1.1 +versus IL-17A + cells gated on CD3
? cells are shown. (B) IL-17A
+versus CD3+ cells (nongated; left) and IL-17A +versus CD4
+ cells gated on CD3 ? cells (right) are shown. Data are representative of three independent experiments.
38IL-17 AND IL-22 PRODUCTION BY SPLENIC LT I -LIKE CELLS | Takatori et al.
tion of IL-17A – p roducing LTi-like cells was determined by FACS. As shown in F ig. 4 (top) the proportion of IL-17 – p roducing LTi-like cells was signifi cantly greater in zymo-san-treated mice compared with control mice. To further establish the ability of LTi-like cells to produce IL-17 in
vivo, we treated Rag2 ? / ? mice with BFA i.v. ( 21 ) and chal-lenged them with zymosan i.p. for 6 h. We observed few IL-17A – p roducing LTi-like cells in control mice, whereas
zymosan-treated mice had a signifi cant increase in IL-17A – p roducing cells ( F ig. 4 , bottom). These results establish that LTi-like cells can rapidly produce IL-17 in vivo when chal-lenged with the product of fungal pathogens.
L
Ti-like cells also produce IL-22
I t has been argued that Th17 cells also preferentially produce IL-22 ( 22 ). Interestingly, we found that treatment with zymo-san induced not only IL-17A but also IL-22 in the sera and
spleens of Rag2
? / ? mice ( F ig. 5 A ). In vitro stimulation with memory CD4 + T cells that selectively produce IL-17 ( 19 ), we found that most of the splenic LTi-like cells express CCR6
( F ig. 3 D ). Approximately half of the CCR6
+ LTi-like cells coexpressed CXCR5 and CCR7 (unpublished data), suggest-ing that the expression of those chemokine receptors on LTi-like cells is heterogeneous, as previously reported ( 20 ). On the other hand, the expression of CXCR3, CCR5, or CCR4, which are preferentially expressed on Th1 or Th2 cells ( 19 ), was low on splenic LTi-like cells (Fig. S6 and not depicted).
L
Ti-like cells produce IL-17 during zymosan-induced infl ammation in Rag2 ? / ? mice
W e next determined whether LTi - l ike cells produced IL - 17 in vivo in an infl ammatory setting. We approached this
problem in two ways: fi rst, we injected Rag2 ? / ? mice with
zymosan i.p. to induce infl ammation and harvested spleno-cytes 2 h later. The cells were then stimulated ex vivo with PMA/Iono and Brefeldin A (BFA) for 2 h, and the propor-
F igure 3. I solated CD4 + C D3 ? C D11c ? B 220 ? LTi-like cells produce IL-17 and constitutively express IL-23R, ROR ? t , AHR, and CCR6. (A) Iso-lated CD4 +CD3 ? CD11c ? B220 ? LTi-like subsets from Rag2 ?/? splenocytes (top) were cultured in the absence (Control) or presence of IL-23 (or with PMA/
Iono) for 24 h, and IL-17A in culture supernatants was measured by ELISA (bottom). Data are means ± SD from duplicate cultures and are representative
of two independent experiments. (B) S tat3 fl /fl or Stat3 fl /fl ; MMTV-Cre splenocytes were cultured in the absence (Control) or presence of IL-23 for 24 h. The
proportion of IL-17A – p roducing LTi-like cells was evaluated by intracellular staining. Data are means ± SD from duplicate cultures and are representative
of four independent experiments ( n = 8). *, P < 0.05. (C) Total RNA was prepared from LTi-like cells isolated from Rag2
?/? spleens. The relative expression levels (/18S) of the indicated mRNA was analyzed by q-PCR and are depicted as fold induction relative to fresh B cells (or fresh naive T cells for AHR mRNA expression). Data are representative of three independent experiments. M-T, memory T cells; N-T, naive T cells. (D) The expression of CCR6 on LTi-like cells of WT splenocytes was analyzed by FACS. The shaded histogram indicates staining with isotype-matched control antibodies. The continuous line histogram indicates the surface expression level of CCR6 on LTi-like cells. Data are representative of three independent experiments. ND, not detected.
JEM VOL. 206, January 19, 2009 39
BRIEF DEFINITIVE REPORT
it is becoming increasingly clear that they are not the only
source of IL-17. Clearly, ? ? T and iNKT cells produce IL-17 to protect against bacterial infection ( 6, 7 ). In addition, a re-cent study provides evidence that IL-23 can drive IL-17 production by innate immune cells in animal models of in-fl ammatory bowel disease ( 23 ).
W e were very impressed by the loss of IL-17 production in Rag2 ? / ? ? c ? / ? mice ( F ig. 1 B ). We initially suspected that
this pointed to production of IL-17 by NK cells but were sur-prised by the failure to see IL-17 production by NK1.1 + C D3
? NK cells ( F ig. 2 A ). However, LTi-like cells are also depen-dent on ? c cytokines for development ( 17 ). Before our stud-ies, it was known that these cells expressed ROR ? t , which was required for their function in LNs ( 16, 18 ). ROR ? t has several important functions, but it is now clear that one func-tion is the regulation of Th17 cell diff erentiation ( 13 ). We confi rmed that splenic LTi-like cells constitutively express ROR ? t ( F ig. 3 C ) and that IL-23 further up-regulated its ex-pression (Fig. S7 C), arguing that this factor is essential in controlling IL-17 and IL-22 production by non – T cells. In-terestingly, fresh LTi-like cells also constitutively expressed more AHR than polarized Th17 cells ( F ig. 3 C ). LTi-like cells also constitutively expressed IL-23R and CCR6 ( F ig. 3,
C and
D ), similar to CCR6 +
I L-23R + human memory CD4 + T cells, which are major producers of IL-17 ( 19, 24 ). Thus, the expression of ROR ? t , AHR, CCR6, and IL-23R seems to be the signature to defi ne IL-17 – and IL-22 – p roducing cells in both adaptive and innate immune cells.
L Ti-like cells are involved in the proper formation of pe-ripheral LNs, the spleen, and gut-associated lymphoid tissue by highly expressing lymphotoxin ? , lymphotoxin ? , and TNF- ? ( 16, 25 ). Our data indicate that splenic and gut LTi-like cells also produce IL-17 and IL-22 ( F igs. 3 A and 5 B ; and Fig. S7, A and D) ( 26 ). It has been recently shown that blocking IL-17 results in disruption of the germinal center –
l ike formation in the spleen, and restoration of the lymphoid microanatomy is dependent on the proliferative accumula-tion of LTi-like cells in SLOs during viral infection ( 27, 28 ). In addition, the cross talk between bacteria, LTi cells, stromal cells, DCs, and B cells seems to be essential for isolated lym-phoid follicle formation in the gut ( 29 ). LTi-like cells might
IL-23 induced more IL-22 production by Rag2 ? / ? spleno-cytes than WT splenocytes ( F ig. 5 B ). Furthermore, isolated LTi-like cells also produced IL-22 ( F ig. 5 B and Fig. S7 D).
Although we observed that NK1.1 +
C D3 ? NK cells failed to produce IL-17A ( F ig. 2 A ), isolated NK cells produced small amounts of IL-22 in response to IL-23 (Fig. S8, available at https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/jem.20072713/DC1). These fi ndings illustrate the potentially distinct modes of reg-ulation for IL-17 and IL-22. T he IL-23 – I L-17 axis has emerged to be important in host defense and in models of autoimmunity such as experi-mental autoimmune encephalomyelitis and infl ammatory bowel disease ( 10, 23 ). Despite the importance of Th17 cells,
F igure 4. Z ymosan induces IL-17 production in splenic LTi-like cells in vivo. (left) Intracellular staining of IL-17A was performed with in vitro conventional staining (top) or in vivo staining (bottom). For conven-tional staining, Rag2
?/? mice were challenged with PBS (Control) or 12.5 mg zymosan i.p. Splenocytes were isolated 2 h later and stimulated with PMA/Iono and BFA for 2 h in vitro. The proportion of IL-17A – p roducing LTi-like cells was evaluated by intracellular staining. For the in vivo stain-ing, Rag2
?/? mice were injected with PBS (Control) or 12.5 mg zymosan i.p. together with 0.25 mg BFA i.v. (reference 22 ). The proportion of IL-17A –
p roducing LTi-like cells in spleens was directly evaluated by intracellular staining. (right) The mean values (horizontal bars) for IL-17A – p roducing LTi-like cells were calculated for in vitro staining (top, n = 6) and in vivo staining (bottom, n = 6). Data are representative of three (top) or two (bottom) independent experiments. ***, P < 0.001.
F igure 5. L Ti-like cells produce high levels of IL-22. (A) Sera and splenocytes were harvested from Rag2
?/? mice 3 h after injection of PBS (Control) or 5 mg zymosan. (left) IL-22 in sera was measured by ELISA. (right) The relative expression levels (/18S) of IL-22 mRNA were determined by q-PCR and are depicted as fold induction relative to control cells. The data are means ± SD from two mice and are representative of two independent experiments.
(B) WT or Rag2 ?/? splenocytes or isolated LTi-like cells were cultured in the presence of IL-23 for 24 h, and IL-22 in culture supernatants was measured
by ELISA. The data are means ± SD from duplicate cultures and are representative of two independent experiments.
40
IL-17 AND IL-22 PRODUCTION BY SPLENIC LT I -LIKE CELLS | Takatori et al.
CD4 + C D3 + C D62L ? C D44 + memory T cells; CD4 + C D3 + C D62L high C D44
low naive T cells; CD19 + B cells, Th17 cells polarized with IL-6, TGF ? -1, and
anti-CD3/-CD28 for 3 d; and CD4 + C D3 ? C D11c ? B 220 ? LTi-like cells.
cDNA was synthesized with the TaqMan Reverse Transcription kit (Ap-plied Biosystems). TaqMan primers and probes for mouse IL-17A, IL-17F, IL-22, IL-23R, IL-12R ? 1, IL-12R ? 2, RORc (for ROR ? t ), IL-17RA, IL-6R ? , CCR6, AHR, GATA3, and 18SrRNA (as endogenous control) were purchased from Applied Biosystems. Samples were analyzed by using a se-quence detection system (ABI PRISM 7700; Applied Biosystems).
T he amounts of cytokines in the culture supernatant were measured us-ing mouse IL-17 Quantikine assay kits (for IL-17A; R & D Systems) and the mouse IL-22 ELISA construction kit (Antigenix America Inc.) according to the manufacturers ’ instructions. IL-23 – m ediated cell stimulations were per-formed at cell concentrations of 4 × 10 6 cells/ml. Samples were measured in duplicate against the standard curve of the assay.
D ata analysis. S tatistical signifi cance was determined by the Student ’ s t test. P < 0.05 was considered to indicate a signifi cant diff erence. O nline supplemental material. F ig. S1 shows IL-17A production by WT
or Rag2 ? / ? splenocytes in the presence of IL-23 with ? c -dependent cyto-kines, anti-CD3/-CD28, or ? -GalCer. Fig. S2 shows that IL-17A – p roducing CD3 ? cells do not express myeloid markers. Fig. S3 shows three populations in CD4 + C D3
? cells from Rag2 ? / ? spleens. Fig. S4 shows that LTi-negative subsets produce minimal IL-17A. Fig. S5 shows IL-17A production by LTi-like cells in the gut. Fig. S6 shows the surface expression of various markers on LTi-like cells. Fig. S7 shows mRNA expression of various factors in LTi-like cells after IL-23 stimulation with IL-7. Fig. S8 shows IL-22 and IL-17A production by isolated NK cells. Online supplemental material is available at https://www.sodocs.net/doc/2c10836149.html,/cgi/content/full/jem.20072713/DC1.
W e thank J. Simone and the fl ow cytometry core, and the animal facility of the NIAMS for excellent advice and technical service. We thank Drs. H. Young and D. McVicar for helpful discussions. We thank Drs. L. Hennighhausen and D. Levy for providing S tat3 fl /fl ;MMTV-Cre mice.
T his research was supported by the Intramural Research Program, NIAMS, NIH. T he authors have no confl icting fi nancial interests. Submitted: 20 December 2007 Accepted: 2 December 2008
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M ATERIALS AND METHODS R ecombinant cytokines and antibodies. M ouse IL-6, IL-12, IL-21, IL-23, and TGF ? -1 were purchased from R & D Systems. Mouse IL-2, IL-4, IL-7, and IL-15 were purchased from PeproTech. Antibodies to CD4 (G K1.5), CD3 (145-2C11), CD28 (37.51), CD11c (HL3), B220 (RA3-6B2), ? c (4G3), IL-7R ? (SB199), Thy1.2 (30-H12), CD44 (IM7), CD62L (MEL-14), CCR5 (C34-3448), CXCR5 (2G8), and IL-17 (TC11-18H10; for IL-17A) were purchased from BD. Antibodies to CD30L (RM153), OX40L (RM134L), and CCR7 (4B12) were purchased from eBioscience. Anti-CCR6 antibody (clone 140706) was obtained from R & D Systems. Both anti-CXCR3 and -CCR4 antibodies were purchased from Abcam. ? -GalCer was obtained from AXXORA, LLC.
M ice. C 57BL/6J WT mice (The Jackson Laboratory), Rag2
? / ? mice, and Rag2 ? / ? ? c ? / ? mice (Taconic) were purchased as indicated. S tat3 fl /fl
mice were bred with mice expressing Cre under the control of the MMTV-LTR
( M MTV-Cre ) to produce S tat3 fl /fl ; MMTV-Cre mice (provided by L.
Hennighhausen [National Institute of Diabetes and Digestive and Kidney Diseases] and D. Levy [New York University, New York, NY]) ( 30 ). All animal experiments were performed according to the National Institutes of Health (NIH) guidelines for laboratory animals and were approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) Ani-mal Care and Use Committee.
I solation of cells and cell culture. S ingle-cell suspensions were prepared
from spleens of healthy 8 – 10-wk-old mice. All cell cultures were performed in RPMI 1640 supplemented with 10% FBS, 2 mM l -glutamine, 5 mM Hepes, 100 U/ml Pen-Strep, and 2.5 μ M 2-ME at 37 ° C for 4, 24, or 48 h. Cells stained with the appropriate antibodies were isolated by fl ow cytomet-ric cell sorting using a Mo-Flo cell sorter (Dako). Whole splenocytes or iso-lated cells were cultured in the presence of 20 ng/ml IL-23, 20 ng/ml IL-4, 10 ng/ml IL-6, 20 ng/ml IL-7, 20 ng/ml IL-15, 20 ng/ml IL-2, 20 ng/ml IL-12, 100 ng/ml IL-21, or 5 ng/ml TGF ? -1. F low cytometric analysis and intracellular cytokine staining. C ells were stimulated for 2 h with 50 ng/ml PMA and 1 μ g /ml Iono, followed by incubation with BFA (GolgiPlug; BD) for an additional 2 h. Cells were fi xed in 4% formyl saline and permeabilized with 0.1% saponin permeabilization buff er after surface staining. PE-conjugated anti – I L-17 antibody was used to detect intracellular cytokine levels (BD). Stained cells with the appropriate antibodies were analyzed on a fl ow cytometer (FACSCalibur; BD). Events were collected and analyzed with FlowJo software (Tree Star, Inc.). To
evaluate production of IL-17A in vivo, Rag2 ? / ?
mice were injected i.p. with 5 or 12.5 mg zymosan (Sigma-Aldrich). Control animals received PBS. To assess in vivo intracellular cytokine levels, 0.25 mg BFA (Sigma-Aldrich) was simultaneously injected i.v., as previously described ( 21 ). R NA isolation and measurement of cytokines. T otal RNA was isolated using TRI z ol reagent (Invitrogen) from freshly isolated
BRIEF DEFINITIVE REPORT
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