One-pot room-temperature conversion of cyclohexane to adipic acid by ozone and UV light

Article abstract of DOI:10.1126/science.1259684

Nitric acid oxidation of cyclohexane accounts for ～95% of the worldwide adipic acid production and is also responsible for ～5 to 8% of the annual worldwide anthropogenic emission of the ozone-depleting greenhouse gas nitrous oxide (N2O). Here we report a N2O-free process for adipic acid synthesis.Treatment of neat cyclohexane, cyclohexanol, or cyclohexanone with ozone at room temperature and 1 atmosphere of pressure affords adipic acid as a solid precipitate. Addition of acidic water or exposure to ultraviolet (UV) light irradiation (or a combination of both) dramatically enhances the oxidative conversion of cyclohexane to adipic acid.

Full text of DOI:10.1126/science.1259684

RESEARCH
| RESEARCH ARTICLES
REFERENCES AND NOTES
HUMORAL IMMUNITY
1
2
.
.
A. Inoue et al., J. Clin. Oncol. 24, 3340–3346 (2006).
E. L. Kwak et al., N. Engl. J. Med. 363, 1693–1703
(
2010).
MAVS, cGAS, and endogenous
retroviruses in T-independent
B cell responses
3
4
.
.
R. Rosell et al., Lancet Oncol. 13, 239–246 (2012).
L. V. Sequist et al., J. Clin. Oncol. 26, 2442–2449
(
A. T. Shaw et al., Lancet Oncol. 12, 1004–1012 (2011).
. C. R. Chong, P. A. Jänne, Nat. Med. 19, 1389–1400
2008).
5.
6
(
2013).
R. Katayama et al., Sci. Transl. Med. 4, 120ra17
2012).
7.
(
1
2
2
1
1
1,4
1
8
9.
1
.
N. Yamaguchi et al., Lung Cancer 83, 37–43 (2014).
J. Tanizaki et al., Clin. Cancer Res. 18, 6219–6226 (2012).
Ming Zeng, Zeping Hu, * Xiaolei Shi, * Xiaohong Li, * Xiaoming Zhan, * Xiao-Dong Li,
1,4
1
1
1
1
Jianhui Wang, Jin Huk Choi, Kuan-wen Wang, Tiana Purrington, Miao Tang,
0. M. J. Niederst, J. A. Engelman, Sci. Signal. 6, re6
2013).
1
2
1
3
Maggy Fina, Ralph J. DeBerardinis, Eva Marie Y. Moresco, Gabriel Pedersen,
Gerald M. McInerney, Gunilla B. Karlsson Hedestam, Zhijian J. Chen, Bruce Beutler †
(
3
3
1,4
1
1
1
1. X. Liu et al., Am. J. Pathol. 180, 599–607 (2012).
2. J. A. Engelman et al., Nat. Med. 14, 1351–1356 (2008).
3. J. A. Engelman et al., Science 316, 1039–1043 (2007).
Multivalent molecules with repetitive structures including bacterial capsular
polysaccharides and viral capsids elicit antibody responses through B cell receptor
1
4. A. C. Faber et al., Proc. Natl. Acad. Sci. U.S.A. 106,
9503–19508 (2009).
5. M. L. Sos et al., Proc. Natl. Acad. Sci. U.S.A. 106, 18351–18356
2009).
1
(BCR) crosslinking in the absence of T cell help. We report that immunization with
1
these T cell–independent type 2 (TI-2) antigens causes up-regulation of endogenous
retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a
mitochondrial antiviral signaling protein (MAVS)–dependent RNA sensing pathway or
reverse-transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a
second, sustained wave of signaling that promotes specific immunoglobulin M production.
Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse
transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings
suggest that ERV and two innate sensing pathways that detect them are integral
components of the TI-2 B cell signaling apparatus.
(
1
1
1
1
6. M. Guix et al., J. Clin. Invest. 118, 2609–2619 (2008).
7. J. Qi et al., Cancer Res. 71, 1081–1091 (2011).
8. L. V. Sequist et al., Sci. Transl. Med. 3, 75ra26 (2011).
9. T. H. Marsilje et al., J. Med. Chem. 56, 5675–5690
(
2013).
0. A. T. Shaw et al., N. Engl. J. Med. 370, 1189–1197
2014).
2
(
2
2
2
2
1. J. L. Marks et al., Cancer Res. 68, 5524–5528 (2008).
2. D. K. Walters et al., Cancer Cell 10, 65–75 (2006).
3. M. W. Karaman et al., Nat. Biotechnol. 26, 127–132 (2008).
4. U. McDermott, R. V. Pusapati, J. G. Christensen, N. S. Gray,
J. Settleman, Cancer Res. 70, 1625–1634 (2010).
pecific antibody production is a hallmark
of the B cell response to antigens. T cell–
dependent (TD) antibody responses typ-
ically elicited by protein antigens require
follicular helper T cells for full B cell acti-
Ficoll (NP-Ficoll) by monitoring anti-NP IgM in the
serum of mice after immunization (2). C57BL/6J
mice mounted a robust NP-specific IgM response
by day 4.5 after immunization, which peaked
around day 14.5 after immunization (Fig. 1A and
fig. S1). Similarly, mice that could not signal via
NLRP3, TLR3, TLR7, TLR9, TLR2, TLR4, CD36,
MyD88, TICAM1, or IRAK4, all nucleic acid–sensing
2
2
2
5. L. F. Hennequin et al., J. Med. Chem. 49, 6465–6488
(2006).
6. S. Bagrodia, I. Chackalaparampil, T. E. Kmiecik, D. Shalloway,
Nature 349, 172–175 (1991).
7. L. Friboulet et al., Cancer Discov. 4, 662–673 (2014).
S
vation, proliferation, and antibody production.
In contrast, T cell–independent (TI) antigens
stimulate antibody production in the absence
of major histocompatibility complex (MHC) class
II–restricted T cell help. TI antigens include the
TI type 1 (TI-1) antigens, which engage Toll-like
receptors (TLRs) in addition to the B cell recep-
tor (BCR), and TI type 2 (TI-2) antigens, which
engage the BCR in a manner that induces exten-
sive crosslinking, leading to BCR activation and
immunoglobulin M (IgM) production. TI-2 anti-
gens are large multivalent molecules with highly
repetitive structures, such as bacterial capsular
polysaccharides and viral capsids (1).
ACKNOWLEDGMENTS
We thank L. H. Park for direction, redirection, and support;
S. R. Vora for continual critical discussion and support;
W. Michaud for providing feeder cells and training for the
development of patient-derived cell lines; and N. Gray for
providing several kinase inhibitors. This study was funded by
support from the NIH R01CA137008 (J.A.E.), R01CA164273
3d/3d
Lps2/Lps2
TLRs (Unc93b1
), or all TLRs (Ticam1
) produced normal levels of NP-
specific IgM on day 4.5 after immunization (Fig.
;
otiose/otiose
Irak4
gt/gt
−/−
(
A.T.S. and J.A.E.), 1U54HG006097-01 (C.H.B.), the Wellcome
1A). In contrast, Tmem173
mice and Mb21d1
Trust (086357 and 102696, C.H.B.), the National Cancer
Institute Lung SPORE P50CA090578 (A.S.C., A.J.I., and J.A.E.),
the Department of Defense (L.V.S. and J.A.E.), Conquer Cancer
Foundation Young Investigator Award (A.S.C.), Uniting Against
Lung Cancer (A.S.C. and A.T.S.), Free to Breathe (A.S.C.),
Lungevity (L.V.S. and J.A.E.), National Foundation for Cancer
Research (A.T.S.), and Be a Piece of the Solution.
J.A.E. is a paid consultant for Novartis, Sanofi-Aventis,
AstraZeneca, Chugai, Amgen, Genentech, GSK, Merck, and
Pfizer, and he holds equity in Gatekeeper Pharmaceuticals,
which has a potential equity interest in T790M inhibitors.
J.A.E. also receives research support from Novartis. J.F.G. is a
paid consultant for Boehringer Ingelheim. A.T.S. is a paid
consultant for Pfizer, Novartis, Ariad, Chugai, Genentech,
Roche, and Ignyta. A.J.I. is a Senior Advisory Board member
and holds equity in Enzymatics Inc. For gene expression
analyses, the raw data are deposited in ArrayExpress
mice, deficient in the cytosolic DNA-sensing path-
way components stimulator of interferon gene
(STING) and cGMP-AMP synthase (cGAS), respec-
tively, exhibited suboptimal IgM responses to
NP-Ficoll on day 4.5 and for up to 30 days after
immunization (Fig. 1A and fig. S1). Mice lacking
mitochondrial antiviral signaling protein (MAVS),
an adaptor for the cytoplasmic RNA-sensing RIG-
I–like helicases, also produced diminished amounts
of NP-specific IgM (Fig. 1A and fig. S1). Antibody
responses to the TI-1 antigen NP-LPS (LPS, lipo-
polysaccharide) (Fig. 1B), and the TD antigen
b-galactosidase (b-gal) encoded by a nonrepli-
cating recombinant Semliki Forest virus (rSFV)
vector (3) (Fig. 1C), were normal in STING-,
cGAS-, and MAVS-deficient mice.
We evaluated marginal zone (MZ) and B-1 B
cell populations in STING-, cGAS-, and MAVS-
deficient mice and found no deficiencies in fre-
quencies or numbers (fig. S2 and supplementary
text), except in the NP-specific populations after
NP-Ficoll immunization (fig. S3). Also, NP-Ficoll
capture by MZ B cells and MZ macrophages was
normal in the mutant mice (fig. S4).
B cell–intrinsic cytosolic DNA and RNA
sensing in the TI-2 antibody response
We tested the requirement for innate immune
sensing pathways in the antibody response to the
model TI-2 antigen 4-hydroxy-3-nitrophenylacetyl-
(
accession number is E-MTAB-783). The normalized data are
available at www.cancerrxgene.org/downloads.
1
Center for the Genetics of Host Defense, University of Texas
Southwestern Medical Center, 5323 Harry Hines Boulevard,
2
Dallas, TX 75390-8502, USA. Department of Pediatrics and
Children's Medical Center Research Institute, and McDermott
Center for Human Growth and Development, University of
Texas Southwestern Medical Center, 5323 Harry Hines
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/346/6216/1480/suppl/DC1
Materials and Methods
Figs. S1 to S16
Tables S1 to S8
Databases S1 to S8
3
Boulevard, Dallas, TX 75390-8502, USA. Department of
Microbiology, Tumor and Cell Biology, Karolinska Institutet,
4
Nobels väg 16, SE-171 77 Stockholm, Sweden. Howard
Hughes Medical Institute, Department of Molecular Biology,
University of Texas Southwestern Medical Center, 5323
Harry Hines Boulevard, Dallas, TX 75390-9148, USA.
*These authors contributed equally to this work. †Corresponding
author. E-mail: Bruce.Beutler@UTSouthwestern.edu
References (28–34)
1
4 April 2014; accepted 21 October 2014
Published online 13 November 2014;
0.1126/science.1254721
We performed adoptive transfer of C57BL/6J,
STING-, cGAS-, or MAVS-deficient splenic and
1
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peritoneal B cells into Rag2−/− mice and immu-
nized recipient mice with NP-Ficoll one day after
transfer. Despite similar reconstitution of the B
cell compartment by donor cells (fig. S5), mice
that received STING-, cGAS-, or MAVS-deficient
B cells produced diminished amounts of NP-
specific IgM on day 4.5 after immunization, as
compared to mice that received C57BL/6J B cells
but not STING-deficient splenic B cells in vitro
(Fig. 2A, fig. S6, and supplementary text). After
NP-Ficoll immunization of C57BL/6J mice, cGAMP
levels were elevated for at least 10 days in NP-
specific B cells relative to levels in non–NP-specific
or naïve B cells (Fig. 2, B and C). In vitro cGAMP
treatment activated B cells from C57BL/6J but not
STING-deficient mice (Fig. 2, D and E), whereas
in vivo cGAMP treatment partially rescued NP-
specific IgM levels in the serum of cGAS-deficient
mice immunized with NP-Ficoll together with
cGAMP (Fig. 2F). Thus, cytoplasmic DNA and
cGAMP are sufficient to activate B cells in a
STING-dependent manner. Human B cells were
also activated by cGAMP treatment in vitro
(Fig. 2G).
(Fig. 1A) suggested that cytosolic RNA may also
activate B cells.
ERV transcription and reverse
transcription in TI-2 antigen–specific
B cells
No exogenous nucleic acid species are known
to be introduced into B cells upon BCR engage-
ment by TI-2 antigens, suggesting that endoge-
nous nucleic acids are responsible for activation
of the cGAS-STING and MAVS pathways. We
hypothesized that RNA and reverse-transcribed
DNA derived from endogenous retroviruses (ERVs)
or retrotransposons might gain access to the B
cell cytoplasm upon BCR signaling. The retro-
viral life cycle entails the transient localization
of RNA and, via reverse transcription, DNA with-
in the cytoplasm (5). Both cGAS-STING (6) and
RIG-I–MAVS pathways (7) have been implicated
in the sensing of HIV-1 and other retroviruses.
We therefore investigated whether ERVs could
be induced by TI-2 immunization.
(
Fig. 1D). These data demonstrate that B cell–
intrinsic MAVS and cGAS-STING signaling
are necessary for antibody responses to TI-2
immunization.
B cell activation by cGAMP
The DNA sensor cGAS binds to cytosolic DNA and
catalyzes the synthesis of cGMP-AMP (cGAMP),
a cyclic dinucleotide that binds and activates
STING, leading to type I interferon production
These findings implicate cytosolic DNA sensed
by the cGAS-STING pathway in B cell activation
by TI-2 antigens. The impaired antibody response
of MAVS-deficient mice immunized with NP-Ficoll
(
4). We found that the presence of DNA in the
cytoplasm was sufficient to activate C57BL/6J,
We measured transcript levels of individual
ERVs in B cells after immunization of C57BL/6J
mice with NP-Ficoll; DNA contamination of the
RNA samples was excluded (fig. S7A). Compared
to non–NP-specific B cells on day 4.5 after immu-
nization, NP-specific B cells displayed increased
expression of multiple ERV mRNAs (Fig. 3A). We
found that total RNA isolated from NP-specific
B cells was significantly more potent than total
RNA from non–NP-specific B cells in activating
cytokine and chemokine gene expression in bone
marrow–derived macrophages (BMDMs) (fig. S7,
B to D). Gene expression induced by NP-specific
B cell total RNA was partially dependent on MAVS
(fig. S7C); ribonuclease treatment abrogated the
effect (fig. S7D). To test the idea that a MAVS-
dependent RNA sensing pathway could be ac-
tivated by ERV RNAs in NP-specific B cells, we
used RIG-I immunoprecipitation followed by re-
verse transcription polymerase chain reaction
(RT-PCR) analysis and found that all tested ERV
mRNA sequences bound to RIG-I molecules in
NP-specific B cells from immunized mice; greater
amounts of ERV mRNA were immunoprecipi-
tated with RIG-I from NP-specific B cells than
from non–NP-specific B cells (Fig. 3B). We also
verified the selective binding of RIG-I to ERV
mRNA versus cellular mRNA (fig. S7E).
Reverse transcription of retroviral RNA occurs
in the cytoplasm and produces cDNA that can
be detected by cGAS (6). Measurement of cyto-
plasmic ERV DNA (fig. S8A) revealed elevated
levels in NP-specific B cells as compared to non–
NP-specific B cells from immunized C57BL/6J
mice (Fig. 3C). Consistent with this finding, the
activity of reverse transcriptase (RT) was up-
regulated in NP-specific relative to non–NP-specific
or naïve B cells (Fig. 3D). We used inhibitors of
RT to determine whether reverse transcription
is necessary for B cell activation and antibody
production in mice immunized with NP-Ficoll.
B cells were cultured in vitro and treated with
NP-Ficoll and the RT inhibitors azidothymidine
(AZT), nevirapine (NVP), and didanosine (ddI).
RT inhibition significantly reduced CD86 and
Fig. 1. Cytosolic DNA and RNA sensing pathways are essential for induction of the TI-2 antibody
response. (A) Serum NP-specific IgM on day 4.5 after immunization with NP-Ficoll. (B) Serum NP-specific
IgM on day 4.5 after immunization with NP-LPS. (C) Serum b-gal–specific IgG on day 14.5 after immuniza-
tion with rSFV-encoded b-gal. (D) Serum NP-specific IgM on day 4.5 after immunization of Rag2− mice
adoptively transferred 1 day before immunization with splenic and peritoneal B cells from donor mice of
the indicated genotypes. Data points represent individual mice. P values were determined by one-way
analysis of variance (ANOVA) and post hocTukey test; in (B) and (C), no significant difference was found
between any mutant genotype and C57BL/6J. Results are representative of two or three independent
experiments.
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GL7 expression in C57BL/6J and MAVS-deficient
NP-specific B cells relative to NP-specific B cells
without RT inhibition (Fig. 3, E and F).
BCR, Btk activation is a defining feature of the
signaling induced by TI-2 antigens (1). Btk-deficient
xid mice failed to produce NP-specific IgM on day
4.5 after immunization with NP-Ficoll (Fig. 4E),
which is consistent with previously reported data
(13). NP-specific B cells from immunized xid
mice expressed similar levels of ERV mRNAs as
non–P-specific B cells from either xid mice or
from control mice (Fig. 4F). In human B cells in
vitro, the BTK inhibitor Ibrutinib blocked both
the expression of activation markers (Fig. 4G)
and the elevation of ERV transcription induced
by anti-IgM (Fig. 4D). These findings demon-
strate that Btk is necessary for the induction of
ERV mRNAs in mouse B cells by TI-2 immu-
nization, and in human B cells by BCR ligation.
Data from several experiments supported a
requirement for NF-kB in the antibody response
to TI-2 immunization, and specifically in the in-
duction of ERV mRNAs. First, NF-kB–dependent
cytokines were up-regulated in NP-specific B cells
from C57BL/6J mice immunized with NP-Ficoll,
whereas non–NP-specific B cells showed cyto-
In vivo, C57BL/6J mice pretreated with AZT
and NVP followed by immunization with NP-
Ficoll showed varying but significant reductions
in ERV DNA levels in the cytoplasm of NP-specific
B cells compared to immunized mice treated with
vehicle (fig. S8B), which is consistent with the
known differential susceptibility of RT enzymes
to distinct RT inhibitors (8, 9). C57BL/6J and
MAVS-deficient mice treated with RT inhibitors
exhibited impaired NP-specific IgM production
relative to immunized vehicle-treated mice (Fig.
kine production similar to that of naïve B cells
panR2
(Fig. 5A). Second, mutation of NEMO (Ikbkg
),
a component of the IKK complex required for
NF-kB activation, abrogated the NP-specific IgM
response of C57BL/6J mice to NP-Ficoll immuni-
zation (Fig. 1A). Third, NP-specific B cells from
NEMO-deficient mice expressed ERV mRNA
levels similar to those of non–NP-specific B cells
(Fig. 5B).
3G). However, no additive impairment of activa-
tion marker expression on NP-specific B cells in
vitro, or of NP-specific IgM production in vivo,
resulted from RT inhibition in STING-deficient
B cells or mice (Fig. 3, E to G). Consistent with
these findings, double knockout of MAVS and
cGAS reduced NP-specific IgM production to
nearly undetectable levels in the serum of im-
munized mice (Fig. 3H). We also immunized
double knockout mice deficient in MAVS and
STING and noted that, although reduced as com-
pared to mice lacking either protein alone, re-
sidual NP-specific IgM production still occurred,
suggesting that cGAS may have functions inde-
pendent of cGAMP production or that alternate
cGAMP receptor(s) remain to be identified (fig.
S9). These data suggest that TI-2 immunization
activates transcription and reverse transcription
to produce ERV mRNAs and cDNAs. These nu-
cleic acids probably then engage the RIG-I–MAVS
and cGAS-STING pathways, leading to signaling
necessary for B cell activation and antigen-specific
antibody production. We note that the poten-
tial involvement in TI-2 antibody responses of
LINE-1 elements, which are also susceptible to
RT inhibition, remains to be tested.
NF-kB is required for ERV induction and
is activated by BCR and MAVS signaling
TI-2 antigens engage the BCR and trigger sig-
naling events, including the activation of Src
kinases, the Tec family kinase Btk, and subse-
quently NF-kB (10), which is known to activate
the transcription of exogenous retroviruses in
other cells (11, 12). Because mutations affecting
the BCR reduce or eliminate B cells, we investi-
gated the involvement of BCR signaling in the
induction of ERVs by measuring ERV mRNA
levels in response to BCR engagement by anti-
IgM in vitro. Anti-IgM treatment increased acti-
vation marker expression (Fig. 4A), caused strong
up-regulation of multiple ERV mRNAs (Fig. 4B),
and elevated cGAMP (Fig. 4C) in C57BL/6J B cells.
Human B cells also up-regulated ERV mRNAs
upon treatment with anti-IgM in vitro (Fig. 4D).
These data indicate that BCR ligation is suffi-
cient to induce ERV transcription in mouse and
human B cells, as well as activation of cGAS in
mouse B cells. MAVS- and cGAS-deficient B cells
failed to fully up-regulate activation markers in
response to anti-IgM (Fig. 4A); these findings are
consistent with MAVS and cGAS activation in-
duced by BCR signaling. Downstream from the
Fig. 2. cGAMP is elevated within antigen-specific B cells after TI-2 immunization and is sufficient
to drive B cell activation in vitro and in vivo. (A) CD86, MHC class II, and CD25 expression, and BrdU
+
–
+
incorporation by GFP or GFP (GFP, green fluorescent protein) splenic CD19 B cells, 36 hours after
gt/gt
transfection with a GFP expression plasmid. N = 3 C57BL/6J mice and 3 Sting
measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in 2 × 10 NP-specific or
non–NP-specific splenic CD19 B cells from C57BL/6J mice on day 4.5 after immunization with NP-Ficoll
mice. (B) cGAMP level
5
+
(N = 3) or in naïve mice (N = 3). Upper panels, chromatograms of cGAMP. Lower panel, cGAMP abundance
normalized to cell number for each sample. (C) Time course of cGAMP levels in NP-specific or non–NP-
specific B cells from C57BL/6J mice (N = 3) immunized with NP-Ficoll. (D and E) CD86 expression (D) or
+
the percentage of BrdU splenic B cells (E) after treatment with cGAMP or vehicle for 2 days in vitro. N = 3
gt/gt
C57BL/6J mice and 3 Sting
mice. (F) Serum NP-specific IgM on day 5 after immunization with NP-
Ficoll plus cGAMP or NP-Ficoll plus vehicle. (G) HLA-DR or CD69 expression by human B cells isolated
from healthy donor peripheral blood and treated with cGAMP or vehicle for 2 days in vitro. MFI, mean
fluorescence intensity. Data points represent individual mice or humans [(B), (F), and (G)]. P values were
determined by one-way [(A), (B), and (D) to (F)] or two-way ANOVA and post hoc Tukey test (C) or
Student’s t test (G). Results are representative of two or three independent experiments.
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Signaling from the BCR, RIG-I–MAVS, and cGAS-
STING pathways all activate NF-kB (10, 14, 15),
and we sought to identify which pathway(s)
drive NF-kB activation needed for the TI-2 anti-
body response. We found that anti-IgM failed
to induce ERV mRNAs in NEMO-deficient B cells
in vitro (Fig. 4B), suggesting that BCR signaling
induces ERV mRNAs via NF-kB. As we have dem-
onstrated, ERV transcripts trigger a second, sus-
tained wave of signaling via the RIG-I–MAVS and
cGAS-cGAMP-STING pathways that may also
require NF-kB to activate B cells and stimulate
antibody production. We evaluated NF-kB acti-
vation by measuring levels of phosphorylated
p65 and p105. In response to anti-IgM, C57BL/6J,
MAVS-, and cGAS-deficient B cells displayed ele-
vated levels of phospho-p65 and phospho-p105
as compared to vehicle-treated cells; however,
MAVS-deficient cells showed a diminished level
of NF-kB activation relative to C57BL/6J cells
(Fig. 5C). Analysis of p65 nuclear translocation
corroborated these findings (Fig. 5D). Similar-
ly, NP-specific B cells from immunized C57BL/6J,
MAVS-, and cGAS-deficient mice displayed ele-
vated phospho-p105 relative to non–NP-specific
B cells; MAVS deficiency prevented NF-kB acti-
vation to the extent observed in C57BL/6J NP-
specific B cells (Fig. 5E). These data suggest that
MAVS signaling but not cGAS-STING signaling
activates NF-kB in response to ERV nucleic acids
Fig. 3. TI-2 antigen immunization induces expression
of ERV mRNA and cDNA that is detected by cytosolic
sensors in antigen-specific B cells. Splenic NP-specific
+
or non–NP-specific CD19 B cells were collected from
B1-8+
C57BL/6J mice (A, C, and D) or Igh
transgenic mice
(B) 4.5 days after immunization with NP-Ficoll (N = 3 mice
per experiment). (A) Transcript levels of the indicated
ERVs measured by reverse transcription quantitative PCR
(RT-qPCR) of mRNA isolated from NP-specific or non–NP-
specific B cells. Data were normalized to glyceraldehyde
phosphate dehydrogenase (GAPDH) mRNA levels in the
same cells. Because of copy number differences, the mag-
nitude of up-regulation of different ERVs cannot be directly
B1-8+
compared in this experiment. (B) Igh
transgenic mice
express a recombined variable region derived from an
+
NP-binding antibody in place of the endogenous 3′ Igh-D element (DQ52)
and the Igh-J elements. RT-qPCR of the indicated ERV mRNAs immunopre-
cipitated with RIG-I from NP-specific or non–NP-specific B cells. Data were
normalized to the level of GAPDH mRNA bound to RIG-I in the same samples,
which represents a nonspecific interaction equivalent in NP-specific and
non–NP-specific cells as shown in fig. S7E. (C) qPCR of ERV DNA in the cyto-
plasmic fraction of NP-specific or non–NP-specific B cells. Data were nor-
malized to GAPDH intronic DNA levels in the same cells. Endogenous ecotropic
murine leukemia virus (eMLV) and mouse mammary tumor virus (MMTV)
were amplified with primers targeting spliced cDNAs; these species probably
represent a minority of the cytoplasmic eMLVand MMTVcDNAs and thus may
not precisely reflect total eMLV and MMTV cDNA levels. (D) RT activity in the
indicated B cells from C57BL/6J mice. (E and F) Splenic CD19 B cells from
mice of the indicated genotypes were treated with NP-Ficoll plus RT
inhibitors (AZT, NVP, and ddI) or NP-Ficoll plus vehicle for 2 days, and CD86
(E) or GL7 expression (F) was measured in NP-specific B cells. N = 3 mice
for each genotype. (G) Serum NP-specific IgM on day 4.5 after NP-Ficoll
immunization of mice pretreated for 3 days with RT inhibitors (AZTand NVP)
or vehicle. RT inhibitor treatment continued after immunization until mea-
surement of serum IgM. (H) Serum NP-specific IgM on day 4.5 after im-
munization with NP-Ficoll. Data points represent individual mice [(D), (G),
and (H)]. P values were determined by Student’s t test [(A) to (C)] or one-
way ANOVA and post hocTukey test [(D) to (H)]. n.d., not detected. Results
are representative of two or three independent experiments.
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induced by BCR engagement. The finding that
NP-specific B cell RNA from immunized mice
transcriptionally activated NF-kB target genes,
including those encoding TNFa, IL-6, MCP-1,
and COX-2, in a MAVS-dependent manner (fig.
S7, C and D) further supports this conclusion.
cGAS-STING and MAVS signaling in
antibody responses to Streptococcus
pneumoniae capsular polysaccharides
We immunized cGAS-, STING-, MAVS-, or cGAS/
MAVS-deficient mice with Streptococcus pneu-
moniae capsular polysaccharides (PSs) 1 and 3
or with the commercial vaccine Pneumovax 23
(
PPV-23), containing 23 S. pneumoniae polysac-
charide antigens. By day 4.5 after immunization,
C57BL/6J mice mounted a PS1- and PS3-specific
IgM response, which was significantly diminished
in cGAS- or MAVS-deficient mice (Fig. 6A). Mice
lacking both cGAS and MAVS displayed a fur-
ther reduction in PS1- and PS-3–specific IgM to
nearly undetectable levels (Fig. 6A). Similarly,
upon immunization with Pneumovax 23, cGAS-
or STING-deficient mice produced diminished
amounts of PPV-23–specific IgM and IgG as com-
pared to C57BL/6J mice (Fig. 6, B and C). Thus,
the requirement for cGAS-STING and RIG-I–
MAVS pathways, likely due to their detection of
ERV nucleic acids, extends to antibody responses
against TI-2 antigens of a clinically important hu-
man pathogen.
Discussion
With respect to ERVs, we hypothesize that the
following events occur in B cells after TI-2 im-
munization: (i) ERV proviruses are transcribed,
and the resulting unspliced and spliced RNAs
are transported to the cytoplasm; (ii) ERV mRNAs
are translated to produce RT(s) (Fig. 3D), Env
glycoprotein(s) (fig. S10A), and possibly other ERV
proteins; (iii) ERV mRNAs are reverse-transcribed
to produce cDNAs by one or more of the RT spe-
cies produced in step (ii); and (iv) ERV mRNAs
and cDNAs present in the cytoplasm activate
RIG-I and cGAS, respectively (fig. S11). Although
retroviruses including HIV-1 and MLV typical-
ly proteolytically process RT from the Gag-Pol
polyprotein during or after virus budding, RT
has also been reported to be active in the pre-
cursor form (16, 17); similar ERV-derived precur-
sors may provide the RT activity we measured
in NP-specific B cell lysates. Overall, our data
support the hypothesis that ERV nucleic acids
induced in the TI-2 antigen–activated B cell are
sufficient to trigger cytosolic innate sensors,
leading to subsequent antibody production by
the same cell (fig. S11). However, despite mul-
tiple experiments supporting the involvement
of ERV nucleic acids, it remains possible that
their presence and activity are incidental to
other component(s) essential for the activa-
tion of MAVS, cGAS, STING, and ultimately
TI-2 antibody responses. Determining whether
ERV nucleic acids represent a necessary com-
ponent of the antibody response will require
comprehensive inactivation of ERV sequences
across the genome.
Fig. 4. cGAMP elevation and ERV transcription are induced by BCR signaling. (A to C) Mouse
splenic B cells were cultured in vitro and stimulated with anti-IgM or vehicle for 22 hours. (A) CD86 and
MHC class II expression. (B) Transcript levels of the indicated ERVs measured by RT-qPCR of isolated
mRNA. Data were normalized to GAPDH mRNA levels in the same cells. N = 3 mice per genotype. (C)
Chromatograms of cGAMP in C57BL/6J B cells measured by LC-MS/MS. N = 3 mice. (D) Human B cells
from healthy donor peripheral blood (N = 3 individuals) were cultured in vitro and stimulated with anti-IgM,
anti-IgM + Ibrutinib, or vehicle for 48 hours.Transcript levels of the indicated human ERVs measured by RT-
qPCR of isolated mRNA. (E) Serum NP-specific IgM on day 4.5 after immunization with NP-Ficoll. (F)
Transcript levels of the indicated ERVs measured by RT-qPCR of mRNA isolated from NP-specific or non–
NP-specific B cells. Data were normalized to GAPDH mRNA levels in the same cells. N = 3 mice per
genotype. (G) Activation marker expression by human B cells treated as in (D), or with 0.06 mM cGAMP for
48 hours. Data points represent individual mice or humans [(A), (E), and (G)]. P values were determined by
Student’s t test [(B) and (D) to (F)] or one-way ANOVA and post hocTukey test [(A) and (G)]. Results are
representative of two independent experiments.
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Fig. 6. TI-2 antibody responses to S. pneumoniae
PS1 and PS3 and to the commercial vaccine
Pneumovax 23 require cytosolic DNA and RNA
sensing pathways. (A) Serum PS1- and PS3-specific
IgM on day 4.5 after immunization with S. pneumoniae
PS1 and PS3. (B and C) Serum PPV-23–specific
IgM (B) and IgG (C) on day 5.5 after immunization
with Pneumovax 23. Data points represent indi-
vidual mice. P values were determined by one-way
ANOVA and post hocTukey test (A) or Student’s t
test [(B) and (C)]. Results are representative of
two or three independent experiments.
Fig. 5. NF-kB is required for ERV induction and is activated by BCR and MAVS signaling. (A and B)
Splenic NP-specific or non–NP-specific CD19 B cells were collected from mice 4.5 days after immunization
with NP-Ficoll. (A) Cytokine expression in the indicated B cells from C57BL/6J mice. (B) Transcript levels
+
of the indicated ERVs measured by RT-qPCR of mRNA isolated from NP-specific or non–NP-specific B
+
–
cells. N = 3 mice. No significant difference was found between NP and NP cells for any ERV tested. (C and
D) Splenic B cells were cultured in vitro and stimulated with anti-IgM or vehicle for 22 hours. (C) Levels of
phospho-p65 (left) and phospho-p105 (right) are shown. N = 3 mice. (D) Levels of p65 in the nuclear
+
autoimmune disease in mice (20, 21). An associ-
ation of human autoimmune diseases with ERVs
has also been postulated (22). Such diseases may
be hypothesized to result from dysregulation of
adaptive rather than strictly innate immune ac-
tivation, in that retroviruses, MAVS, cGAS, and
STING serve as components of the normal TI-2 B
cell signaling apparatus.
fraction of cells. (E) Levels of phospho-p105 in splenic NP-specific and non–NP-specific CD19 B cells or
naïve B cells on day 6 after NP-Ficoll immunization. N = 3 mice per genotype. Data points represent
individual mice [(A) and (D)]. P values were determined by Student’s t test (B) or one-way ANOVA and
post hoc Tukey test [(A) and (C) to (E)]. Results are representative of two independent experiments.
The appropriation of ERV genes for host im-
munity has often been observed in defense
against exogenous retroviruses, as exemplified
by Fv1 (18) and Fv4 (19). Whereas the host re-
cruits a single ERV-derived gene to counteract
one particular step in the life cycle of an exog-
enous retrovirus, however, it activates a wide
range of ERVs in antigen-specific B cells that
then serve to trigger intracellular nucleic acid
sensors necessary for the TI-2 antibody response.
The fate of these ERV nucleic acids remains un-
known. We found in NP-specific B cells from im-
munized mice that the expression of endogenous
murine leukemia virus–specific Env glycoprotein
was enhanced (fig. S10A), as was RT activity de-
tected in the supernatants (fig. S10B). Why ERV
proteins are produced and how they might come
to exist outside the cell during the TI-2 antibody
response are open questions.
REFERENCES AND NOTES
1.
J. J. Mond, A. Lees, C. M. Snapper, Annu. Rev. Immunol. 13,
55–692 (1995).
6
2
3
.
Materials and methods are available as supplementary
materials on Science Online.
A. S. Hidmark et al., J. Virol. 80, 7100–7110 (2006).
X. Cai, Y. H. Chiu, Z. J. Chen, Mol. Cell 54, 289–296 (2014).
S. P. Goff, Nat. Rev. Microbiol. 5, 253–263 (2007).
D. Gao et al., Science 341, 903–906 (2013).
.
.
4
Recent reports suggest that aberrant cellular
accumulation of endogenous retroelement cDNA
and its recognition by cytosolic sensors lead to
5.
6
.
7. M. Solis et al., J. Virol. 85, 1224–1236 (2011).
SCIENCE sciencemag.org
19 DECEMBER 2014 • VOL 346 ISSUE 6216 1491

RESEARCH
8
.
R. M. Ruprecht, L. G. O’Brien, L. D. Rossoni, S. Nusinoff-Lehrman,
Nature 323, 467–469 (1986).
S. D. Young et al., Antimicrob. Agents Chemother. 39,
19. H. Ikeda, F. Laigret, M. A. Martin, R. Repaske, J. Virol. 55,
768–777 (1985).
20. G. B. Beck-Engeser, D. Eilat, M. Wabl, Retrovirology 8,
91-4690-8-91 (2011).
21. D. B. Stetson, J. S. Ko, T. Heidmann, R. Medzhitov, Cell 134,
587–598 (2008).
22. N. Tugnet, P. Rylance, D. Roden, M. Trela, P. Nelson, Open
Rheumatol. J. 7, 13–21 (2013).
supplementary materials. This work was supported by generous
donations from the Lyda Hill Foundation and the Kent and
JoAnn Foster Family Foundation; and by NIH grants P01 AI070167
and U19 AI100627 (to B.B.), R01 AI093967 (to Z.J.C.), and
R01 CA157996 (to R.D.).
9.
2602–2605 (1995).
1
0. M. E. Moreno-García, K. M. Sommer, A. D. Bandaranayake,
D. J. Rawlings, Adv. Exp. Med. Biol. 584, 89–106 (2006).
1. J. Alcamí et al., EMBO J. 14, 1552–1560 (1995).
2. A. Roulston, R. Lin, P. Beauparlant, M. A. Wainberg, J. Hiscott,
Microbiol. Rev. 59, 481–505 (1995).
1
1
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/346/6216/1486/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S11
Table S1
References (23–32)
ACKNOWLEDGMENTS
13. W. N. Khan et al., Immunity 3, 283–299 (1995).
14. X. D. Li et al., Science 341, 1390–1394 (2013).
We thank L. Evans and F. Malik for the generous gift of the
83A25 antibody; P. Jurek for expert assistance in preparing the
figures; and the Children’s Medical Center Research Institute
Flow Cytometry Facility at the University of Texas Southwestern
Medical Center for assistance with cell sorting. The data presented
in this paper are tabulated in the main paper and in the
15. Y. M. Loo, M. Gale Jr., Immunity 34, 680–692 (2011).
16. S. Crawford, S. P. Goff, J. Virol. 53, 899–907 (1985).
17. F. Lori et al., AIDS Res. Hum. Retroviruses 4, 393–398 (1988).
18. S. Best, P. Le Tissier, G. Towers, J. P. Stoye, Nature 382,
23 June 2014; accepted 6 November 2014
10.1126/science.1257780
826–829 (1996).
◥
complements existing strategies employing either
chemical modifications—for example, of pho-
tosynthetic reaction centers (19)—or nonlinear
laser methods (20–22) that use ultrashort pulsed
laser light in the ultraviolet (UV) or visible spec-
tral regions.
REPORTS
ELECTRON TRANSFER
Control of ET processes using IR excitation is
underpinned by a strong theoretical framework
pertaining to vibronic interactions in the non-
adiabatic (weakly electronically coupled) electron
tunneling medium. This framework introduces
multiple vibronic coupling pathways between
electron donor and acceptor, whereby the inter-
ference between the electrons propagating through
the different pathways directs ET. Theoretical
analyses of vibronic tunneling pathways in model
molecules by Beratan and co-workers predicted
that in inelastic ET reactions, where a tunneling
electron may interact and exchange energy with
a bridge-localized vibrational mode, IR driving of
pathway-specific vibrations can effectively label
and select the ET path (23–25). Such experiments
would thus offer the means to control ET reaction
outcomes using external IR excitation.
Toward control of electron transfer
in donor-acceptor molecules by
bond-specific infrared excitation
1
1
1,2
2
Milan Delor, Paul A. Scattergood, Igor V. Sazanovich, Anthony W. Parker,
2
1
Gregory M. Greetham, Anthony J. H. M. Meijer,
2
1
Michael Towrie, * Julia A. Weinstein *
Electron transfer (ET) from donor to acceptor is often mediated by nuclear-electronic
vibronic) interactions in molecular bridges. Using an ultrafast electronic-vibrational-
(
vibrational pulse-sequence, we demonstrate how the outcome of light-induced ET can be
radically altered by mode-specific infrared (IR) excitation of vibrations that are coupled to
the ET pathway. Picosecond narrow-band IR excitation of high-frequency bridge vibrations
in an electronically excited covalent trans-acetylide platinum(II) donor-bridge-acceptor
system in solution alters both the dynamics and the yields of competing ET pathways,
completely switching a charge separation pathway off. These results offer a step toward
quantum control of chemical reactivity by IR excitation.
Indeed, vibrational excitation with broad-
−1
band (~120 cm ) IR pulses that perturbed the
H-bonding network linking donor and acceptor
molecules was shown to slow down an inter-
molecular charge-separation process and decrease
its yield by ~1.8% (26). These elegant experiments
successfully demonstrated the viability of using IR
pulses to change the course of an ET reaction.
Our alternative approach to direct ET uses a
he fundamental process of electron trans-
fer (ET) is central to many light-driven
reactions. Light-induced ET in molecules
starts by excitation to a so-called Franck-
Condon electronic excited state, in which
pendence of ET rates on the vibrational quantum
number of the reactant state (5). Taken together,
these phenomena offer an enticing strategy to
influence ET reactions by exciting vibrations
coupled with the ET process, thus perturbing
nuclear-electronic (vibronic) interactions, which
are known to mediate ET in systems ranging
from heterogeneous assemblies to complex bio-
logical architectures (6–15). If achieved, this strategy
provides a means to alter transition probabilities
and ultimately redefine the nature and the yields
of products generated in ET processes.
The challenge of directing ET is particularly
complex owing to the ultrafast time scales and
multicoordinate energy landscape of this class of
reactions (3). Because the energies of molecular
vibrations lie in the IR region, low-energy IR
light can be used to target specific vibrations and
potentially affect photoreactivity—an approach
successfully implemented in gas-phase photo-
chemistry (16–18). With regard to manipulating
photoinduced ET processes, the use of IR light
T
−1
nonzero vibrational levels are usually populated
1–3). The excess vibrational energy is rapidly
transform-limited narrowband (~12 cm , 1.5 ps)
(27) IR pulse to achieve mode-specific excitation
of intramolecular vibrations directly coupled to
ET in a covalently bound donor-bridge-acceptor
(DBA) molecular triad. The designed DBA triad
1, based on a platinum(II)-trans-acetylide motif
(
lost as the excited state relaxes and nuclear re-
arrangements begin to occur. ET may occur on
time scales commensurate with the depopu-
lation of these “vibrationally hot” states, typi-
cally 10− to 10 s (4), allowing ET to proceed
from such nonthermalized states. Previous in-
frared (IR) spectroscopy studies with vibrational
state resolution have shown a significant de-
14
−10
(28, 29), PTZ-CH
-Ph-CꢀC-Pt-CꢀC-NAP (30), en-
2
forces directionality of ET between a powerful
electron donor, phenothiazine (PTZ), and an elec-
tron acceptor, naphthalene-monoimide (NAP)
(Fig. 1A). 1 represents an ideal substrate for IR
modulation as the high-frequency CꢀC stretch-
1
Department of Chemistry, University of Sheffield, Sheffield S3
2
−1
7
HF, UK. Central Laser Facility, Research Complex at Harwell,
ing vibrations of the bridge at ~2100 cm have
Rutherford Appleton Laboratory, Harwell Science and Innovation
Campus, Science and Technology Facilities Council, Chilton,
Oxfordshire OX11 0QX, UK.
Corresponding author. E-mail: mike.towrie@stfc.ac.uk (M.T.),
julia.weinstein@sheffield.ac.uk (J.A.W.)
previously been identified as the ET reaction
coordinate in related Pt(II) acetylides (31) and
are sufficiently separated in energy and space
*
−1
from the nearest intense IR absorption [~1700 cm ,
1
492 19 DECEMBER 2014 • VOL 346 ISSUE 6216
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MAVS, cGAS, and endogenous retroviruses in T-independent B cell
responses
Ming Zeng et al.
Science 346, 1486 (2014);
DOI: 10.1126/science.346.6216.1486
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