Myeloid-specific tristetraprolin deficiency in mice results in extreme lipopolysaccharide sensitivity in an otherwise minimal phenotype.
ABSTRACT Tristetraprolin (TTP) is a mRNA-destabilizing protein that binds to AU-rich elements in labile transcripts, such as the mRNA encoding TNF, and promotes their deadenylation and degradation. TTP-deficient (knockout [KO]) mice exhibit an early-onset, severe inflammatory phenotype, with cachexia, erosive arthritis, left-sided cardiac valvulitis, myeloid hyperplasia, and autoimmunity, which can be prevented by injections of anti-TNF Abs, or interbreeding with TNF receptor-deficient mice. To determine whether the excess TNF that causes the TTP KO phenotype is produced by myeloid cells, we performed myeloid-specific disruption of Zfp36, the gene encoding TTP. We documented the lack of TTP expression in LPS-stimulated bone marrow-derived macrophages from the mice, whereas fibroblasts expressed TTP mRNA and protein normally in response to serum. The mice exhibited a minimal phenotype, characterized by slight slowing of weight gain late in the first year of life, compared with the early-onset, severe weight loss and inflammation seen in the TTP KO mice. Instead, the myeloid-specific TTP KO mice were highly and abnormally susceptible to a low-dose LPS challenge, with rapid development of typical endotoxemia signs and extensive organ damage, and elevations of serum TNF levels to 110-fold greater than control. We conclude that myeloid-specific TTP deficiency does not phenocopy complete TTP deficiency in C57BL/6 mice under normal laboratory conditions, implying contributions from other cell types to the complete phenotype. However, myeloid cell TTP plays a critical role in protecting mice against LPS-induced septic shock, primarily through its posttranscriptional regulation of TNF mRNA stability.
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ABSTRACT: Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative and migratory capacity of endothelial cells, as well as control of leukocyte trafficking. Endothelial dysfunction is an early step in vascular inflammatory diseases such as atherosclerosis, diabetic vascular complications, sepsis-induced or severe virus infection-induced organ injuries. The expressions of inflammatory cytokines and vascular adhesion molecules induced by various stimuli, such as modified lipids, smoking, advanced glycation end products and bacteria toxin, significantly contribute to the development of endothelial dysfunction. The transcriptional regulation of inflammatory cytokines and vascular adhesion molecules has been well-studied. However, the regulation of those gene expressions at post-transcriptional level is emerging. RNA-binding proteins have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level in microRNA-dependent or independent manners. This review summarizes the latest insights into the roles of RNA-binding proteins in controlling vascular endothelial cell functions and their contribution to the pathogenesis of vascular inflammatory diseases.Science China. Life sciences 08/2014; 57(8):836-844. · 1.51 Impact Factor
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ABSTRACT: Zinc finger protein 36, C3H type-like 1 (ZFP36L1) is one of several Zinc Finger Protein 36 (Zfp36) family members, which bind AU rich elements within 3' untranslated regions (UTRs) to negatively regulate the post-transcriptional expression of targeted mRNAs. The prototypical member of the family, Tristetraprolin (TTP or ZFP36), has been well-studied in the context of inflammation and plays an important role in repressing pro-inflammatory transcripts such as TNF-α. Much less is known about the other family members, and none have been studied in the context of infection. Using macrophage cell lines and primary alveolar macrophages we demonstrated that, like ZFP36, ZFP36L1 is prominently induced by infection. To test our hypothesis that macrophage production of ZFP36L1 is necessary for regulation of the inflammatory response of the lung during pneumonia, we generated mice with a myeloid-specific deficiency of ZFP36L1. Surprisingly, we found that myeloid deficiency of ZFP36L1 did not result in alteration of lung cytokine production after infection, altered clearance of bacteria, or increased inflammatory lung injury. Although alveolar macrophages are critical components of the innate defense against respiratory pathogens, we concluded that myeloid ZFP36L1 is not essential for appropriate responses to bacteria in the lungs. Based on studies conducted with myeloid-deficient ZFP36 mice, our data indicate that, of the Zfp36 family, ZFP36 is the predominant negative regulator of cytokine expression in macrophages. In conclusion, these results imply that myeloid ZFP36 may fully compensate for loss of ZFP36L1 or that Zfp36l1-dependent mRNA expression does not play an integral role in the host defense against bacterial pneumonia.PLoS ONE 01/2014; 9(10):e109072. · 3.53 Impact Factor
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ABSTRACT: For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the resolution of inflammation.PLoS Genetics 06/2014; 10(6):e1004368. · 8.52 Impact Factor
The Journal of Immunology
Myeloid-Specific Tristetraprolin Deficiency in Mice Results in
Extreme Lipopolysaccharide Sensitivity in an Otherwise
Lian-Qun Qiu,* Deborah J. Stumpo,* and Perry J. Blackshear*,†,‡
Tristetraprolin (TTP) is a mRNA-destabilizing protein that binds to AU-rich elements in labile transcripts, such as the mRNA
encoding TNF, and promotes their deadenylation and degradation. TTP-deficient (knockout [KO]) mice exhibit an early-onset,
severe inflammatory phenotype, with cachexia, erosive arthritis, left-sided cardiac valvulitis, myeloid hyperplasia, and autoimmu-
nity, which can be prevented by injections of anti-TNF Abs, or interbreeding with TNF receptor-deficient mice. To determine
whether the excess TNF that causes the TTP KO phenotype is produced by myeloid cells, we performed myeloid-specific disruption
of Zfp36, the gene encoding TTP. We documented the lack of TTP expression in LPS-stimulated bone marrow-derived macro-
phages from the mice, whereas fibroblasts expressed TTP mRNA and protein normally in response to serum. The mice exhibited
a minimal phenotype, characterized by slight slowing of weight gain late in the first year of life, compared with the early-onset,
severe weight loss and inflammation seen in the TTP KO mice. Instead, the myeloid-specific TTP KO mice were highly and
abnormally susceptible to a low-dose LPS challenge, with rapid development of typical endotoxemia signs and extensive organ
damage, and elevations of serum TNF levels to 110-fold greater than control. We conclude that myeloid-specific TTP deficiency
does not phenocopy complete TTP deficiency in C57BL/6 mice under normal laboratory conditions, implying contributions from
other cell types to the complete phenotype. However, myeloid cell TTP plays a critical role in protecting mice against LPS-induced
septic shock, primarily through its posttranscriptional regulation of TNF mRNA stability.
with highly conserved sequences and spacing. Initially discovered
as a gene that could be induced rapidly and transiently by the
is now well established that TTP is a mRNA-destabilizing protein
that binds to AU-rich elements (AREs) in its target mRNAs, such
as that encoding TNF (5, 6). TTP-deficient mice appear normal at
birth, but soon develop a complex inflammatory phenotype con-
sisting of cachexia, dermatitis, conjunctivitis, destructive arthritis,
myeloid hyperplasia, and autoimmunity (7), which resembles in
some respects the inflammatory syndrome seen in TNF overpro-
duction transgenic mouse models reported earlier (8, 9).
As the first-described and best-studied TTP target transcript, the
TNF mRNA serves as a prototype target for posttranscriptional
regulation of gene expression by TTP. The TNF mRNA contains
several closely spaced and overlapping copies of the nonamer
The Journal of Immunology, 2012,
ristetraprolin (TTP) is the prototype member of a small
family of RNA-binding proteins that are characterized by
two nearly identical tandem CCCH zinc finger domains
UUAUUUAUU, the optimal TTP-binding motif, in its 39-un-
translated region (39-UTR). After direct binding to these sequence
elements in the 39-UTR of the TNF transcript, TTP then promotes
the removal of its poly(A) tail, followed by its accelerated deg-
radation (5, 10). The functional relevance of the TNFARE in mice
was established by Kontoyiannis et al. (11), who demonstrated
that genetic removal of this element led to stabilization of the TNF
transcript and a systemic inflammatory phenotype similar to, but
more severe than, that seen in the TTP knockout (KO) mice. The
inflammatory phenotype in TTP-deficient mice could be prevented
by repeated injections of anti-TNF Ab, or interbreeding with TNF
receptor-deficient mice (7, 12). These findings highlight the fact
that TNF itself is by far the most important mediator in the
pathogenesis of the mouse TTP-deficiency syndrome, although
other TTP target transcripts have been identified subsequently,
such as those encoding GM-CSF (13), immediate early response-3
(14), polo-like kinase 3 (15), and others.
TNF can be produced by a wide variety of cell types, of both
hematopoietic and nonhematopoietic lineages, including lympho-
cytes, mast cells, and stromal cells (11, 16, 17). Previous studies
from our group have identified macrophages as one of the im-
portant cellular sources that contribute to the TNF overproduction
in TTP KO mice (18). TNF can also promote its own expression;
innate immune system stimuli such as LPS can thus stimulate
TNF expression both primarily and secondarily through TNF itself
(17). In this setting, TTP deficiency and the resulting stabilization
of the TNF mRNA result in disruption of the normal feedback
mediated by TTP, and the state of chronic TNF excess that
characterizes the TTP-deficient mice (7, 19). Transplantation of
bone marrow from TTP KO mice into RAG-22/2immunodefi-
cient mice reproduced the complete TTP deficiency inflammatory
phenotype after a lag period of several months (18), demonstrating
that one or more cell type(s) of hematopoietic origin, probably
*Laboratory of Signal Transduction, National Institute of Environmental Health
Sciences, National Institutes of Health, Research Triangle Park, NC 27709;
†Department of Medicine, Duke University Medical Center, Durham, NC 27710; and
‡Department of Biochemistry, Duke University Medical Center, Durham, NC 27710
Received for publication December 30, 2011. Accepted for publication March 10,
This work was supported by the Intramural Research Program of the National Insti-
tute of Environmental Health Sciences, National Institutes of Health.
Address correspondence and reprint requests to Dr. Perry J. Blackshear, 111 TW
Alexander Drive, Research Triangle Park, NC 27709. E-mail address: Black009@niehs.
The online version of this article contains supplemental material.
Abbreviations used in this article: ARE, AU-rich element; BMDM, bone marrow-
derived macrophage; ES cell, embryonic stem cell; KO, knockout; M-TTP KO,
myeloid-specific TTP-deficient; MEF, mouse embryonic fibroblast; TTP, tristetrap-
rolin; 39-UTR, 39-untranslated region; WT, wild-type.
cells other than lymphocytes, were responsible for triggering the
inflammatory responses that eventually led to the development of
the full TTP deficiency syndrome.
In this study, we hypothesized that specific deficiency of TTP in
myeloid cells would completely recapitulate the early-onset, severe
inflammatory phenotype that is characteristic of the TTP KO mice
(7). To test this hypothesis, we generated a floxed Zfp36 mouse
line, and crossed these mice with mice expressing the Cre
recombinase transgene under the control of the Lysozyme M
promoter. Surprisingly, mice with myeloid-specific deletion of
TTP (myeloid-specific TTP-deficient [M-TTP KO] mice) did not
recapitulate the early-onset, severe inflammatory phenotype of the
TTP KO mice. Instead, these mice exhibited increased suscepti-
bility to a low-dose LPS challenge, with rapid development of an
endotoxemia syndrome with extensive organ damage that was
associated with dramatic increases in circulating TNF. Our results
demonstrate that myeloid-specific TTP deficiency has much less
effect than complete TTP deficiency on C57BL/6 mouse growth
and development under normal laboratory conditions. However,
myeloid cell TTP appears to be critical for the protection of mice
from LPS-induced septic shock, primarily through its ability to
regulate TNF expression at posttranscriptional steps.
Materials and Methods
Generation of M-TTP KO mice
Heterozygous mice with a conditional floxed Zfp36 allele were generated by
gene targeting in embryonic stem (ES) cells by Xenogen Biosciences
and a 4.6-kb MluI–SalI fragment isolated from BAC clone RP23-342K19
were used as 59 and 39 homologous regions, respectively (Fig. 1A). A 1.9-kb
loxP-flanked MluI–SalI fragment was generated for the conditional KO re-
gion, resulting in a vector designed to delete exon 2, including the poly-
adenylation signal, of Zfp36. A loxP-flanked neorexpression cassette was
inserted into intron 1 between the 59-homologous arm and the conditional
KO region. The loxP-flanked Neo expression cassette, and the diptheria
toxin-A gene fragment expression cassette in the vector, were used for
positive and negative selection in ES cells, respectively. The composition of
the final vector was confirmed by restriction digestion and end sequencing.
C57BL/6 ES cells were then electroporated with 30 mg SwaI-linearized
targeting construct and selected in G418 (200 mg/ml). ES clones with ho-
mologous recombination were identified by Southern blotting using a 59
Southernconfirmationused theNeo probe and 39 and 59 external probes, and
the successful integration of the third loxP site was identified by PCR
screening. Two positive ES clones confirmed for homologous recombination
were selected for transient Cre transfection using electroporation for Cre
recombinase-mediated excision of the neorexpression cassette, and two
targeted clones with deletion of the neorexpression cassette were identified
and confirmed upon expansion by PCR analysis. Blastocyst injections were
6N Tac wild-type (WT) females. The floxed Zfp36 mice were maintained by
following primer pair (primer 1, 59-GAA CCC TCT CTC GAT CGG GGA
TAC-39; primer 2, 59-GGATGG AGT CCG AGT TTATGT TCC AA-39),
yielding amplicons of 514 bp for the floxed Zfp36 allele, and 327 bp for the
WTallele, as distinguished by agarose gel electrophoresis.
M-TTP KO mice were achieved by crossing the loxP-flanked Zfp36
mice (Zfp36flox/flox) with mice expressing Cre recombinase under the control
of the murine M lysozyme promoter, which is specific for cells of the
myeloid lineage (LysMcre) (20). Homozygous LysMcre mice on a C57BL/
6 background were purchased from The Jackson Laboratory (Bar Harbor,
ME), and mated with the heterozygous Zfp36flox/+animals, to generate
heterozygous conditional TTP mice with LysMcre (LysMcre/Zfp36flox/+).
Heterozygous matings, LysMcre/Zfp36flox/+mice crossed with Zfp36flox/+
mice, were employed to generate M-TTP KO mice and their littermate
control mice. Double-floxed mice without LysMcre (+/Zfp36flox/flox), mice
with a WT TTP allele, but carrying LysMcre (LysMcre/Zfp36+/+), and WT
mice were used as WT controls. The integration of loxP or the Cre
recombinase transgene into the mouse genome did not cause any evident
changes in morphology or responses to stimuli, in both cell and intact
mouse experiments (data not shown).
For PCR genotyping, genomic DNA from tail clips was extracted, as
described (21). The LysMcre transgene was detected using three primers,
as follows: 59-CCC AGA AAT GCC AGATTA CG-39, 59-CTT GGG CTG
CCA GAATTT CTC-39, and 59-TTA CAG TCG GCC AGG CTG AC-39,
as per The Jackson Laboratory’s recommendations. The myeloid-specific
deleted Zfp36 allele was examined in multiplex reactions using forward
primer 3 (59-CTG GCT GGA AAT GAG AGA GG- 39) and reverse pri-
mers 2 (as described above) and 4 (59-CAC CCC TTA CGC CAG AAC
TA-39), which amplified the WT (683 bp), floxed (870 bp), or deleted
Zfp36 alleles (769 bp; Fig. 3A, 3B). All of the animal breeding and other
procedures were approved by the Institutional Animal Care and Use
Committee of National Institute of Environmental Health Sciences.
Culture of bone marrow-derived macrophages
Eight- to 12-wk-old male mice were euthanized by CO2inhalation, and
bone marrow cells were isolated from the femurs, as described previously
(18). After overnight culture in T25 flasks, nonadherent bone marrow cells
were collected and cultured in RPMI 1640 medium supplemented with
10% heat-inactivated FBS (HyClone, Logan, UT), 25 mM HEPES, 2 mM
glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Life Technolo-
gies, Invitrogen), as well as 30% (v/v) L929 cell-conditioned medium.
Culture medium was replaced by fresh medium every 3 d. Adherent
macrophage monolayers were obtained within 8–10 d; these cells were
.99% positive for Mac-1+Ab staining and negative for Gr-1, as deter-
mined by flow cytometric analysis, identifying them as mature macro-
phages. Differentiated bone marrow-derived macrophages (BMDM) were
then harvested by gently scraping the cells from the dishes using a rubber
policeman and seeded onto 100-mm petri dishes for experiments. Cells
were subjected to serum starvation with RPMI 1640 medium containing
1% FBS for at least 20 h before stimulation with 1 mg/ml LPS (serotype
055:B5; Sigma-Aldrich, St. Louis, MO) for the times indicated.
Mouse embryonic fibroblast culture and stimulation
LysMcre/Zfp36flox/+mice were crossed with +/Zfp36flox/floxmice to obtain
M-TTP KO embryos. Littermate Zfp36flox/floxmice without LysMcre were
used as controls. Primary cultures of mouse embryonic fibroblasts (MEF)
were prepared from embryos at day 15.5 of gestation (E15.5) in which
E0.5 was the date of detection of the vaginal plug. The genotypes for
individual fetuses from each litter were determined by evaluation of tail
DNA from each embryo (7). MEF were maintained at 37˚C (5% CO2) in
DMEM (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen), 100
U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine; MEF at
passages 2–4 were used for the experiments described in this study. For
serum deprivation before stimulation, cells at ∼70–80% confluence were
washed once in serum-free DMEM and then incubated in DMEM con-
taining 0.5% (v/v) FBS for at least 16 h (14, 15). The cells were then
stimulated with 10% (v/v) FBS (HyClone) for the indicated times.
Cytosolic extracts and whole-cell lysates were prepared in radioimmu-
noprecipitation assay buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1%
Nonidet P-40, 5 mM EDTA, and 5% glycerol) or hypertonic lysis buffer
(100 mM Tris-HCl [pH 8.0], 0.5 M NaCl, 5 mM EDTA, 1.25% Nonidet P-
Indianapolis, IN), respectively, cleared by centrifugation, and quantified
using the Bradford assay (Bio-Rad, Hercules, CA). Denatured lysates were
separated on 10–20% Criterion Tris-HCl precast gels (Bio-Rad) and
transferred onto nitrocellulose membranes. Blots were incubated at 4˚C
overnight with rabbit antiserum raised against a recombinant mouse TTP-
maltose–binding protein fusion protein (14, 22), followed by incubation
with HRP-conjugated goat anti-rabbit (Fab9)2(Pierce, Rockford, IL). Im-
mune complexes were detected using SuperSignal West Pico chemilumi-
nescent substrate (Pierce).
LPS challenge in mice
31 g were used. The mice were housed in a pathogen-free animal facility at
25˚C and were illuminated by 12:12-h light-dark cycles. The mice were
provided with standard rodent chow and water ad libitum. Experiments
were carried out in accordance with National Institutes of Health guide-
lines for animal treatment, housing, and euthanasia. Mice were challenged
by the i.p. injection of LPS at a low dose of 0.5 mg/kg (O11:B4; Sigma-
Aldrich) in 200 ml sterile saline, with total amounts ranging from 12 to
15.5 mg LPS per mouse, and monitored for general condition and survival.
Body temperatures were taken with an instant infrared ear thermometer
(VET-TEMP Model VT-150; Advanced Monitors, San Diego, CA). Serial
The Journal of Immunology5151
blood samples were collected through retro-orbital sinus bleeding at 0, 1.5,
3, and 6 h after LPS injection. At 10 h after the LPS challenge, the mice
were euthanized, and serum and organs were collected for chemistry
analyses and histological examinations. Serum levels of aspartate amino-
transferase, alanine aminotransferase, blood urea nitrogen, and lactate
dehydrogenase were determined to assess the extent of muscle, kidney, and
Histological and immunohistochemical analysis
Tissues were fixed in 10% phosphate-buffered formalin and processed using
standard procedures. The 5-mm paraffin tissue sections were cut and stained
with H&E. Samples of bones and paws were decalcified with RDO rapid
decalcifying solution (Apex Engineering Products, Aurora, IL) for 1 h at
room temperature, before being processed and embedded like the others.
For immunostaining, sections were incubated in 3% H2O2to inactivate
endogenous peroxidases, followed by Ag retrieval with heat and pressure
in citrate buffer (Biocare Medical). Endogenous biotin was blocked with
avidin-biotin blocking reagents (Vector Laboratories, Burlingame, CA).
Sections were then incubated with anti-CD45 Ab (GeneTex, San Antonio,
TX) for 60 min at a 1:250 dilution, followed by peroxidase-conjugated
streptavidin SS labeling (Biogenex Laboratories, San Ramon, CA) for
30 min. Immunolabeled Ag–Ab complexes were visualized using di-
aminobenzidine. The sections were lightly counterstained with hematox-
ylin before analysis.
Flow cytometric analysis
Spleens and bone marrow cells were removed from freshly euthanized 8- to
12-wk-old female mice. Single-cell suspensions were prepared in ice-cold
PBS by passing minced tissue through a 100-mm nylon cell strainer, and
then 2 3 106cells were stained with the respective Abs for 20 min at 4˚C
after RBC lysis and preincubation with anti-FcgR (2.4G2; BD Pharmin-
gen) for 15 min. Abs against CD3ε FITC (145-2C11), CD19 allophyco-
cyanin Cy7 (1D3), Gr-1 PE Cy7 (RB6-8C5), and CD11b AF 647 (Mac-1)
were purchased from BD Pharmingen. Anti-CD4 PE (GK1.5) and anti-
CD8a PE Cy5 (53-6.7) were from eBioscience, and anti-F4/80 R-PE (CI:
A3-1) was from Serotec. Stained cells were acquired on a LSR II flow
cytometer using FACSDiva (BD Biosciences) software and analyzed with
FlowJo software (Tree Star, Ashland, OR). Cell debris and dead cells were
excluded from the analysis based on scatter signals and/or propidium io-
Measurement of cytokine levels in sera and culture
Concentrations of IL-1b and IL-6 in supernatants from primary macro-
phages were determined by ELISA using the OptEIA mouse kits (BD
Biosciences), as per the manufacturer’s instructions. Quantification of TNF
levels in serum and culture supernatants was carried out using capture and
biotinylated anti-mouse TNF Abs from BioLegend (San Diego, CA).
Mouse rTNF from eBioscience was used as a standard. Peroxidase-
conjugated avidin (BioLegend) was applied before the colorimetric de-
velopment with the 39,39,5,59-tetramethylbenzidine liquid substrate system
(Sigma-Aldrich). The reactions were stopped with 5 N hydrogen chloride,
and the absorbance at 450 wavelengths (OD 450) was measured with the
Tecan Infinite 200 Pro microplate reader with a 570 wavelength (OD 570)
set as reference.
RNA extraction, real-time RT-PCR, and determination of
mRNA half life
Total cellular RNA from differentiated macrophages was isolated using the
GE Healthcare Illustra RNAspin MiniRNA Isolation Kit, according to the
manufacturer’s instructions (GE Healthcare). Residual genomic DNA was
removed by on-column digestion with RNase-free DNase I. To determine
the decay rates of cytokine transcripts, actinomycin D (5 mg/ml; Sigma-
Aldrich) was added to block transcription after BMDM were stimulated
with LPS (1 mg/ml) for 1 or 4 h. Total RNA was isolated at the indicated
times over a 2-h time course after addition of actinomycin D. TTP and
cytokine transcript levels were determined by real-time RT-PCR. First-
strand cDNAs were synthesized using oligo(dT)12–18primers and Super-
Script III Reverse Transcriptase (Invitrogen). Real-time PCR was performed
using SYBR Green and the ABI Prism 7900 Sequence Detection System
(Applied Biosystems). Primers were designed and compared with the cur-
rent mouse genome reference sequence using BLAST to ensure that no
cross-reactivity with other genes would occur. Results were normalized
against the b-actin transcript as an internal control, and were then used to
calculate expression levels according to the DD cycle threshold method
(23). All data were expressed in terms of fold change relative to the
unstimulated WT sample, which was set as 1, unless otherwise specified.
The primers were validated for their amplification efficiency and specificity
prior to being used in the study; sequences are available upon request.
Data are presented as means 6 SEM. Statistical differences between WT
and mutant groups were determined by the two-sided, unpaired Student t
test. A p value ,0.05 was considered to be significant.
M-TTP KO mice
To examine the effects of myeloid cell TTP deficiency, we gen-
erated a conditional TTP KO mouse line (Fig. 1A) and crossed it
with a Cre mouse line that expresses the Cre recombinase trans-
gene driven by the Lysozyme M promoter (LysMcre). Expression
of this transgene is limited to myeloid cells, including monocytes,
mature macrophages and granulocytes, and a small proportion of
dendritic cells (20). Cre activity should result in the deletion of
exon 2 and the 39-UTR of Zfp36 in the myeloid lineage; exon 2
encodes the tandem zinc finger domain that is responsible for the
RNA-binding activity of TTP and its family members (Fig. 1A).
Morphological phenotype of the M-TTP KO mice
Growth curves. The M-TTP KO mice were viable, fertile, and born
with the expected Mendelian frequency (data not shown). The
growth curves of both male and female M-TTP KO mice under
normal laboratory conditions were almost identical to those of their
littermate controls until the growth curves began to diverge in
females at ∼6.5 mo of age and in males after ∼9 mo (Fig. 1B).
These growth curves are in marked contrast to those from the total
TTP KO mice, which begin to diverge sharply from those of their
WT littermates as early as 3–4 wk of age (7) (Fig. 1B). It should
be noted that, although the TTP KO growth data shown in Fig. 1B
are from ongoing natural history data from our mouse colony, both
the M-TTP KO mice and their littermate controls, and the TTP
KO mice shown in Fig. 1B, are on a pure C57BL/6 background,
and are therefore directly comparable. Although the growth curves
of both the male and female M-TTP KO mice eventually diverged
modestly, but significantly from those of their WT littermates (Fig.
1B), none of these mice has ever become sick enough to require
euthanasia, as is almost universal with the TTP KO mice within
the first several months of life (data not shown).
Histology. Histological analyses of 8-mo-old M-TTP KO mice
revealed very few of the abnormal findings typical of total TTP KO
mice at this age or earlier, including severe polyarticular arthritis,
loss of body fat, myeloid hyperplasia, dermatitis, left-sided cardiac
valvulitis, and glomerular mesangial thickening (7, 24). An ex-
ception to this rule included slightly swollen front paws in some of
the mice; one of four mice analyzed pathologically at this age had
accompanying mild interphalangeal arthritis and inflammation
(Supplemental Fig. 1). This is in contrast to the severe arthritis
seen in all TTP KO mice at or before 7 mo of age (7). In addition,
foci of mild inflammation and fibrosis were observed in the
myocardium of three of four M-TTP KO mice that were examined
at 8 mo of age; these lesions were absent in control mice (data not
shown). Occasional enlargement of lymph nodes and spleens was
noted in some cases in the M-TTP KO mice; overall, there were
modest increases in the average spleen weights (0.103 6 0.004 g,
n = 7) and the ratios of spleen weight/body weight (0.506 6 0.019
g, n = 7) in 3- to 4-mo-old female mutant mice when compared
with their controls (0.083 6 0.002 for the spleen weights and
0.407 6 0.010 for the ratios of spleen weight/body weight in the
WT controls, n = 8; p = 0.0012 and p = 0.0003 for WT versus M-
TTP KO mice, respectively).
5152 MYELOID-SPECIFIC TRISTETRAPROLIN DEFICIENCY
Blood counts. Analysis of peripheral blood from 8-mo-old M-TTP
KO mice revealed normal percentages of blood cells and WBC
differential counts (Fig. 2A). FACS analyses of splenocytes and
bone marrow cells collected from 3-mo-old M-TTP KO mice and
their corresponding controls exhibited normal percentages of
CD3+T lymphocytes and CD19+B lymphocytes, and the T cell
subsets CD4+Th and CD8+cytotoxic cells in the spleens and bone
marrows of M-TTP KO mice were similar to those of WT mice
(Fig. 2B). Likewise, the percentages of CD11b+macrophages,
Gr-1+granulocytes, and CD11b+Gr-1+cells, a subset of immature
myeloid cells, were found to be comparable with those of controls
Studies of cells derived from the M-TTP KO mice
To confirm the TTP deficiency in myeloid cells from the M-TTP
KO mice, we cultured BMDM from these mice. Successful
elimination of the normal Zfp36 alleles in the BMDM from the M-
TTP KO mice was confirmed in genomic DNA isolated from these
cells (Fig. 3A). Both the floxed and WT Zfp36 alleles were barely
detectable, and instead, the disrupted fragment resulting from Cre-
mediated excision was present (Fig. 3A). We then stimulated these
cells with LPS and measured TTP mRNA and protein. The basal
TTP transcript levels in the M-TTP KO BMDM were ∼100-fold
lower than those seen in littermate WT control cells (Fig. 3B,
inset). LPS stimulation of the cells resulted in almost no detect-
able increase in TTP mRNA levels in the M-TTP KO cells,
whereas there was a robust response to LPS in the WT cells (Fig.
3B). Similarly, there was no detectable TTP protein in the M-TTP
KO cells after LPS stimulation, despite readily detected LPS-
induced immunoreactive TTP in the WT cells (Fig. 3C). These
data demonstrate the essentially complete absence of TTP mRNA
and protein expression in BMDM derived from the M-TTP KO
The expression of the other two TTP family members, Zfp36l1
and Zfp36l2 mRNAs, was also examined in these LPS-treated
BMDM. There was modest induction of both Zfp36l1 and
Zfp36l2 transcripts in both WT and M-TTP KO cells within 1 h
after LPS challenge; in both cases, these levels then rapidly de-
creased below basal (Supplemental Fig. 2). For both transcripts,
levels in the KO cells were slightly, but significantly higher than
those found in the WT cells at some time points, but these modest
changes were apparently not sufficient to compensate for the TTP
We next examined the cell-type specificity of the Cre-mediated
version of the strategy used for the generation of the floxed TTP and M-
TTP KO mice. Illustrative maps are provided for the WT Zfp36 locus
(WT), targeting vector, targeted allele, floxed Zfp36 allele (flox) after Cre-
mediated recombination, and the deleted Zfp36 locus (Δ) in the targeted
cells. The open boxes represent coding exons; closed triangles indicate
loxP sites; n = NotI; K = KpnI; Bs = BsiWI; Sp = Spe1; RV = EcoRV; E =
EcoRI; M = MluI. The primers (P1–P4) used for genotyping are indicated
by arrows. (B) Growth curves of M-TTP KO mice, and their littermate
controls, in comparison with those of total TTP-deficient (TTP KO) mice,
were monitored at weekly intervals. Data are presented as mean 6 SEM of
6–49 mice per time point in the M-TTP KO and WT groups, and of 3–11
mice in the TTP KO groups. *p , 0.05,#p , 0.01 for the M-TTP KO
versus WT mice of each sex. As discussed in the text, the M-TTP KO mice
and their littermate controls were studied in parallel, but the data from the
total TTP KO mice are from an ongoing natural history study in our mouse
colony; all mice were on the same genetic background, C57BL/6.
Generation of M-TTP KO mice. (A) Shown is a schematic
KO mice. (A) Complete blood cell counts and WBC differential counts
were performed in peripheral blood from M-TTP KO mice (n = 4) and
littermate WT controls (n = 7, consisting of 1 +/Zfp36+/+, 3 LysMcre/
Zfp36+/+, and 3 +/Zfp36flox/floxmice) at 8 mo of age. Results shown are
mean 6 SEM, and statistical significance is indicated (#p , 0.01). (B)
Flow cytometric analysis of cellular composition in the spleens and bone
marrow cells from 12-wk-old M-TTP KO female mice and WT mice.
CD4+and CD8+cells were analyzed after gating on CD3+cells. The
numbers in quadrants indicate the percentage of cells in each quadrant.
Data are representative of four to five mice in each group. The WT control
used in this study consisted of 1 +/Zfp36+/+and 3 LysMcre/Zfp36+/+mice.
Hgb, Hemoglobin; Lymph, lymphocyte; Mono, monocytes; Neut, neutro-
phil; Plts, platelets.
Hematological and immunophenotypic analyses of M-TTP
The Journal of Immunology5153
for their highly inducible TTP expression in response to multiple
stimuli, including serum (14). MEF were derived from E15.5 M-
TTP KO embryos and their littermate controls. In the cells from
the M-TTP KO mice, the floxed Zfp36 allele was detectable by
PCR of genomic DNA, but the potentially deleted Zfp36 fragment
mediated by LysM-Cre excision was not detectable (Fig. 3D).
Serum stimulation of these cells after overnight serum deprivation
produced virtually identical increases in TTP mRNA levels in the
cells from the WT and M-TTP KO mice (Fig. 3E). Similarly, the
induction of immunoreactive protein in response to LPS was
virtually identical in cells from the WTand M-TTP KO mice (Fig.
3F). These data demonstrate that the myeloid-specific Cre trans-
gene did not excise the floxed allele to any significant extent in
MEF, a prototype nonmyeloid cell type.
Stability of TNF mRNA and protein production in M-TTP KO
To determine whether macrophages derived from the M-TTP KO
mice exhibited similar patterns of TNF transcript stabilization to
those seen in the total TTP KO mice, we measured TNF mRNA
levels and stability in BMDM after LPS stimulation. In control
BMDM, TNF mRNA levels increased rapidly after LPS stimula-
tion, and returned to near basal levels after 24 h (Fig. 4A). Mutant
BMDM exhibited increases in TNF mRNA levels that were 2- to
3-fold higher than control at all times between 1 and 6 h after LPS
stimulation (Fig. 4A). We next examined the stability of the TNF
mRNA in control and M-TTP KO macrophages after adding ac-
tinomycin D 1 and 4 h after LPS stimulation. As expected, the
times to 50% decay of the TNF mRNA were 3-fold greater in the
mutant macrophages than in the corresponding control cells when
actinomycin D was added after either 1 or 4 h of LPS treatment
(Fig. 4B, 4C). Specifically, the average t1/2of the TNF mRNA
were 55 and 67 min in the M-TTP KO macrophages 1 and 4 h
after LPS, respectively, compared with 18 and 23 min in WT cells
(p = 0.03 and 0.001, respectively).
We also measured TNF protein released by BMDM under the
same circumstances. In the WT cell cultures, protein levels reached
a maximum at ∼8 h after LPS stimulation and remained at that
level for the duration of the experiment (Fig. 4D, Supplemental
Fig. 3). In the M-TTP KO cells, there was a ∼6-fold increase in
the medium TNF after 8- and 24-h LPS stimulation compared with
control (Fig. 4D). The differences in protein concentration were
statistically significant (p , 0.05) at all time points between 2 and
Expression of other proinflammatory cytokines
IL-6 and IL-1b are two other proinflammatory cytokines that are
thought to play critical roles in LPS responses, and posttran-
scriptional regulation has been reported to be one of the important
determinants of their expression (25, 26). We therefore examined
MEF (D) was performed in the indicated cells after overnight serum starvation. Primers P2, P3, and P4, as indicated in Fig. 1A, were used to detect the
presence of the floxed Zfp36 allele (870 bp; flox) and WT allele (683 bp; wt). Cre-mediated deletion of a portion of Zfp36 in macrophages resulted in
a novel PCR product of 769 bp (Δ, A). The WT (wt) and deleted alleles (Δ) were not detected in MEF (D). (B and E) WTand M-TTP KO macrophages (B)
and MEF (E) were stimulated with LPS (1 mg/ml, B) or serum (10%, E) for the indicated times, and TTP mRNA levels were analyzed using real-time RT-
PCR. Each point represents the mean of TTP mRNA levels 6 SEM from four independent experiments. The WT control consisted of macrophages isolated
from seven mice of three different genotypes [2 +/Zfp36+/+, 2 LysMcre/Zfp36+/+, and 3 +/Zfp36flox/floxmice; (B)], and of fibroblasts from double-floxed
littermate embryos (E). The full circles and solid line are from the WT controls; the open circles and dashed line are from the TTP KO mice. The inset in (B)
shows the difference in TTP mRNA levels at time 0 in the WTand M-TTP KO (KO) macrophages before LPS stimulation. Each bar represents the mean 6
SEM of values from four independent experiments (p , 0.0001). (C and F) TTP protein levels were measured in similar experiments, as determined by
Western blotting of cytosolic (C) or total (F) lysates prepared from BMDM (C) or MEF (F) after LPS or serum stimulation, respectively, for the indicated
times. The main species of immunoreactive TTP at an approximate maximum Mrof 47,000 were detected in the WT cells; there was no detectable TTP
protein in the KO cells. These results are representative of four independent experiments. Lower panel, Shows immunoreactive b-actin, which served as an
internal gel-loading control.
Targeted TTP deletion in BMDM, but not in MEF, from M-TTP KO mice. (A and D) PCR analysis of genomic DNA from BMDM (A) and
5154MYELOID-SPECIFIC TRISTETRAPROLIN DEFICIENCY
the expression patterns of IL-6 and IL-1b mRNAs, and their
turnover rates, in LPS-stimulated BMDM isolated from the M-
TTP KO mice and their littermate controls. After LPS stimulation,
there was robust induction of both IL-6 mRNA and protein in
BMDM; however, these levels were comparable between the M-
TTP KO macrophages and control cells (Fig. 5A). In the case of
IL-1b mRNA, there was a slightly higher basal level of the IL-1b
transcript in the TTP-deficient BMDM (2.01 6 0.41 compared
with 1.03 6 0.04 in WT cells; p , 0.01), and there was a tendency
for higher levels of this transcript between 2 and 6 h after LPS in
the mutant cells, which did not reach statistical significance
(p = 0.20–0.81; Fig. 5A). IL-1b protein was not detectable in the
culture supernatants from either mutant or WT cells until 24 h
after LPS treatment, when a modest, 1.5-fold decrease in IL-1b
accumulation was seen in the TTP-deficient macrophages (p ,
0.05; Fig. 5B).
When the stabilities of the IL-6 and Il-Lb transcripts were
examined in BMDM after adding actinomycin D at 1 and 4 h after
LPS stimulation, both transcripts remained stable over the 2-h
time course of the experiment in both control and TTP-deficient
cells (Fig. 5C, 5D). Thus, under these experimental conditions, we
were unable to detect changes in IL-6 and IL-1b mRNA stability
in the TTP-deficient BMDM.
Sensitivity of the M-TTP KO mice to LPS
To examine the sensitivity of the M-TTP KO mice to an LPS
At this dose in normal mice, LPS stimulates proinflammatory
cytokine expression, but typically does not provoke significant
signs of endotoxemia (27). Unexpectedly, the otherwise normal-
appearing M-TTP KO mice at 3 mo of age exhibited dramatic and
typical signs of endotoxemia, including lethargy, tachypnea,
piloerection, and marked decreases in body temperature. This
became notable as early as 1.5 h following LPS injection; the
temperature readings dropped below the lower detection limit
after 3 h in all the M-TTP KO mice tested (Fig. 6A). In contrast,
neither the WT mice receiving low-dose LPS or vehicle, nor the
M-TTP KO mice receiving the vehicle alone, exhibited such
endotoxemia signs (Fig. 6A). Per Institutional Animal Care and
Use Committee guidelines, all of the mice were then euthanized at
10 h after LPS administration.
We next examined the serum levels of TNF, as the critical
mediator of endotoxin shock, as well as the product of the best-
studied TTP target transcript. Elevated basal TNF levels (142 6
32 pg/ml) were seen in serum samples from the 3-mo-old M-TTP
KO mice before LPS exposure; this was significantly higher than
the average value from control mice (8 6 6 pg/ml; p = 0.0087).
After the LPS injection, a parallel kinetic pattern of TNF induc-
tion was seen in both M-TTP KO and control mice, with peak
responses at 1.5 h after LPS, followed by a return to baseline after
6 h (average peak values were 67 6 13 ng/ml in the M-TTP KO
mice versus 612 6 113 pg/ml in the controls at 1.5 h; p = 0.0002;
Fig. 6C). The serum levels of TNF in the M-TTP KO mice were
remarkably higher than those in the control mice at all time points
examined, with the peak response in the mutants ∼110-fold higher
than that seen in the WT mice.
Necropsy of these M-TTP KO mice 10 h after LPS adminis-
tration revealed enlarged spleens and dilated small intestines, with
widespread inflammatory cell infiltrates in liver, kidney, and lung
(Fig. 7); the infiltrating leukocytes were predominantly localized
either inside the lumens of vessels or surrounding the vasculature,
suggesting the mobilization and migration of these cells during
endotoxemia. Parenchymal infiltration was also remarkable in the
mutant mice, reflected by scattered foci of hepatic necrosis,
glomerular hypercellularity, and pulmonary alveolitis (Fig. 7,
Supplemental Fig. 4). The M-TTP KO mice treated with saline,
and the control mice treated with either LPS or saline, exhibited
minimal abnormal pathology (Fig. 7, Supplemental Fig. 4). Serum
TNF mRNA expression in BMDM from M-TTP KO mice and their littermate controls was examined by real-time RT-PCR after stimulation with LPS (1
mg/ml) for various times. (B) Levels of TNF accumulating in the supernatants of macrophage cultures stimulated as in (A) at different time points were
measured by ELISA. (C and D) TNF mRNA decay rates were examined after adding actinomycin D (5 mg/ml) into BMDM cultures previously stimulated
with LPS for 1 h (C) or 4 h (D). TNF transcript abundance was determined by real-time RT-PCR, normalized to b-actin mRNA levels, and expressed as
fractions of their abundance in the respective LPS-treated samples prior to the addition of actinomycin D. Results shown in (A)–(D) are means 6 SEM of
values from four independent experiments consisting of four M-TTP KO mice and seven control mice. The WT control in (A)–(C) consisted of cells derived
from 2 +/Zfp36+/+, 2 LysMcre/Zfp36+/+, and 3 +/Zfp36flox/floxmice; the WT control in (D) consisted of cells derived from 1 +/Zfp36+/+, 2 LysMcre/Zfp36+/+,
and 4 +/Zfp36flox/floxmice. *p , 0.05, **p , 0.01,#p , 0.001 when comparing values from WT versus M-TTP KO cells.
Deletion of macrophage TTP results in the increased stability of TNF transcripts and enhanced TNF production after LPS stimulation. (A)
The Journal of Immunology5155
samples from the M-TTP KO mice exhibited a 5-fold increase in
serum alanine aminotransferase, a 2.2-fold increase in blood urea
nitrogen, and a 3-fold increase in lactate dehydrogenase compared
with control mice (Fig. 6B), reflecting liver and kidney dysfunc-
The major findings of this study are that myeloid-specific defi-
ciency of TTP results in no overt phenotype in mice during the
first several months of life under normal laboratory conditions, in
contrast to the early-onset, severe inflammatory syndrome char-
acteristic of total TTP deficiency; and the M-TTP KO mice were
nonetheless highly susceptible to activation of the innate immune
system bya lowdose ofLPS thathad littleor noeffecton littermate
WTmice. Ouroverallconclusionsare thatTTPdeficiencyinoneor
more other cell types is likely to be required for the full-blown
syndrome to develop in this strain of mice, but that regulated
expression of myeloid cell TTP is necessary for the prevention of
septic shock in the setting of infection with Gram-negative bacteria
and perhaps other innate immune system stimuli.
Concerning the first major finding, the principal hypothesis
behind this study was that specific deficiency of TTP in myeloid
cells would completely recapitulate the early-onset, severe in-
flammatory phenotype that is characteristic of the TTP KO mice
(7). This was based on several pieces of supporting evidence,
including the facts that macrophages and, to a lesser extent, other
myeloid cells are the major physiological sources of TNF (16,
17); that TTP-deficient macrophages strikingly overproduce TNF
after stimulation with LPS (18, 19); and that total bone marrow
transplantation can recapitulate the entire, severe TTP deficiency
syndrome after a lag period of several months, suggesting that
hematopoietic cells other than lymphocytes are the primary
drivers of the complete syndrome (18). However, the specific KO
of TTP in myeloid cells using a conditional Zfp36 allele and Cre
driven by the LysM promoter did not lead to the same phenotype
as seen in the total TTP KO mice. Instead, under normal labora-
the proinflammatory cytokines IL-6 and IL-1b are not
affected in BMDM derived from M-TTP KO mice
after LPS stimulation. (A) The induction of IL-6 and
IL-1b mRNA was analyzed by real-time RT-PCR in
LPS (1 mg/ml)-stimulated BMDM isolated from WT
and M-TTP KO mice. (B) Shown are levels of these
cytokines in the culture supernatants of BMDM from
M-TTP KO and WT mice at various times after LPS
addition. ND, not detectable. *p , 0.05, values from
WT versus M-TTP KO cells. (C and D) Decay rates of
IL-6 and IL-1b transcripts were analyzed in M-TTP
KO BMDM after 1-h (C) and 4-h (D) stimulation with
LPS. Actinomycin D (5 mg/ml) was added to BMDM
after LPS treatment to determine the decay rates of
IL-6 and IL-1b transcripts. Results shown are means 6
SEM of values from four independent experiments,
consisting of four M-TTP KO mice and seven control
mice. The WT control consisted of cells derived from
2 +/Zfp36+/+, 2 LysMcre/Zfp36+/+, and 3 +/Zfp36flox/flox
mice. Statistical differences, determined by Student t
test, are indicated (*p , 0.05, M-TTP KO versus WT).
Inducible expression and decay rates of
5156 MYELOID-SPECIFIC TRISTETRAPROLIN DEFICIENCY
tory conditions and on the same genetic background, the M-TTP
KO mice were remarkably healthy, with modest weight loss and
other signs of TTP deficiency first appearing, in mild form, after 6
mo or more of age.
One potential explanation for these surprising results might be
inadequate excision of Zfp36 by this Cre driver in myeloid cells.
However, this was tested in primary macrophages derived from
these mice at the level of the genome, as well as with basal and
LPS-stimulated TTP mRNA and protein production. In all cases,
the data confirm the essentially complete excision of this gene in
the myeloid cells, as well as the complete loss of gene expression.
This was in the setting of totally normal serum-induced TTP ex-
pression in fibroblasts, a nonmyeloid cell type. Thus, we cannot
ascribe the mildness of this syndrome to inadequate Zfp36 dele-
tion in the targeted cells.
Because the entire TTP deficiency syndrome can be transplanted
with whole bone marrow (18), these findings suggest that TTP
deficiency in one or more other types of hematopoietic cells is also
necessary for the full syndrome to evolve in the setting of myeloid
TTP deficiency. It may be possible to uncover the roles of other
cell types using various types of coculture experiments, using, for
example, WT or TTP KO lymphocytes cultured with the M-TTP
KO macrophages under conditions that would allow or prevent
direct cell-cell contact. Alternatively, it might be possible to re-
capitulate the severe TTP deficiency syndrome using conditional
TTP deletion in hematopoietic stem cells; this could eliminate
nonhematopoietic cell types from playing major primary roles in
the pathogenesis of this syndrome.
An informative previous experiment was the use of the TNF
DARE allele, which leads to a greatly stabilized TNF mRNA and
a severe inflammatory syndrome that in many respects resembles
the syndrome of total TTP deficiency (11). A conditional version
to a low-dose endotoxin challenge. (A) Mean body temperatures are
shown for M-TTP KO mice (KO, n = 7) and their littermate controls (WT,
n = 7; 4 +/Zfp36+/+, 2 LysMcre/Zfp36+/+, and 1 +/Zfp36flox/floxmice) after
the i.p. injection of either 0.5 mg/kg LPS or an identical volume of saline
(n = 2 in each group). Body temperatures were measured by an infrared
ear thermometer when the mice were anesthetized before bleeding at each
time point. The thermometer used had a limited reading range of 93–109˚
F, and the temperatures for all of the M-TTP KO mice injected with LPS,
but none of the others, were below the limit of detection within 6 h of the
LPS injection. (B) Serum levels of tissue damage indicators in M-TTP KO
mice 10 h after the LPS injection. Shown are average serum levels of
alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood
urea nitrogen (BUN), and lactate dehydrogenase (LDH) in the M-TTP KO
and WT mice 10 h after the LPS injection (n = 7 each group); *p , 0.05,
**p , 0.01. (C) Kinetics of TNF induction were analyzed in serum from
the M-TTP KO mice (open symbols, n = 7) and littermate WT controls
(solid symbols, n = 7) after i.p. injection of LPS (0.5 mg/kg). Shown are
mean values 6 SEM at each time point. TNF levels in the M-TTP KO
mice were significantly higher than those seen in the control mice at all
time points, with p = 0.0002 at 1.5 h, and p , 0.05 for the rest of the time
TTP deletion in myeloid cells results in extreme sensitivity
KO mice 10 h after a low-dose LPS injection. (A) Representative photo-
micrographs demonstrate CD45-positive inflammatory cell infiltrates
(brown) in the liver (upper panel, original magnification 320), kidney
(middle panel, original magnification 320), and lung (bottom panel,
original magnification 320), in M-TTP KO mice (right panels) and their
littermate control mice (left panels). Neighboring sections stained with
H&E are shown in Supplemental Fig. 4. (B) H&E-stained spleen sections
from a M-TTP KO mouse (right panel) and its littermate control (left
panel) (original magnification 34). Acute splenic enlargement was evident
in the M-TTP KO mice, with the influx of plasma cells, neutrophils, and
macrophages into the red pulp and disruption of large blood vessel walls.
Extensive inflammatory tissue damage in 3-mo-old M-TTP
The Journal of Immunology5157
of this allele was combined with the myeloid-specific Cre driver
used in the current study; in contrast to the severe, early-onset
inflammatory disorder caused by total deletion of the TNFARE in
both heterozygous and homozygous mice, restricting expression
of the DARE allele to myeloid cells resulted in mice that were
overtly normal for at least the first 4 mo of life (28). After this
point, they began losing weight and ultimately developed a milder
version of the inflammatory bowel disease that occurs in an earlier
onset, more severe form in the heterozygous or homozygous TNF
DARE mice. Because we believe that most aspects of the TTP
deficiency syndrome are caused by excessive production of TNF,
this study is another example of how stabilization of the TNF
mRNA in myeloid cells alone is apparently not sufficient to lead to
illness in the absence of other cell types with the same defective
The second major finding of this study is that normal, inducible
TTP expression in myeloid cells is indispensable for protecting
mice against an LPS challenge. This conclusion is based on the fact
that the M-TTP KO mice exhibited extreme sensitivity to a low-
dose LPS exposure that led to typical signs and laboratory find-
ing of endotoxemia, under conditions in which control mice were
unaffected. The extreme sensitivity of these mice to LPS was
presumably caused, at least in part, by the high serum concen-
trations ofTNFthat occurredafter the LPSinjections.These inturn
could be attributed to the high levels of TNF secreted by macro-
phages and other myeloid cells, as demonstrated in culture of
primary BMDM; this resulted from increased TNF mRNA levels
and stability in the TTP-deficient cells. These results are supported
by previous findings that macrophages and neutrophils were the
predominant cellular sources of systemic TNF after LPS in mouse
models with selective TNF deletion in myeloid cells or lympho-
cytes (29). Similar observations to ours have been reported in
the TNFΔAREmice, in which elevated levels of circulating TNF,
and a ∼50% mortality rate, were seen after a normally sublethal
LPS challenge (11).
In contrast withthe apparent central roleof TTPinregulatingthe
stability of TNF mRNA, and subsequent TNF secretion, in this
study, we found little evidence to support a major effect of TTP on
the expression of two other critical mediators of endotoxemia, IL-
1b and IL-6, at least under these experimental conditions. Tran-
scripts of both cytokines contain AU-rich elements, and a recent
study reported that IL-6 and IL-1b transcripts were elevated in the
livers of TTP KO mice (26). Furthermore, the combination of
hypoxia and LPS, with subsequent TTP activation, was able to
destabilize IL-6 transcripts in Raw 264.7 cells (26, 30). However,
in BMDM derived from the M-TTP KO mice, we found little
evidence of instability for these transcripts after LPS stimulation
and actinomycin D treatment, in contrast to the obvious effects
of TTP deficiency on the unstable TNF transcript. There are nu-
merous studies linking other ARE-binding proteins to the stabil-
ities of these transcripts (25, 31), and it may be that they are more
important to physiological regulation of IL-1b and IL-6 transcript
stability and expression than TTP.
Our studies did not address a parallel role for TTP in the reg-
ulation of TNF mRNA translation. Previous studies have suggested
that the TNF mRNA ARE can function as a translation-repressive
element, in addition to its well-known role as a transcript-
destabilizing element (11). In TTP-deficient macrophages, we
have consistently found that TNF protein expression in the TTP-
deficient cells in response to LPS is greater than expected from the
changes in TNF mRNA stability alone (19). In addition, we have
also found that translation of other TTP target messages is greater
in the setting of TTP deficiency, under conditions in which tran-
script levels are largely unchanged (W.S. Lai and P.J. Blackshear,
unpublished data). This would be in keeping with previous studies
with other ARE-binding proteins that are thought to act at least in
part by inhibiting TNF mRNA translation through direct ARE
Our studies describe the extreme sensitivity of the M-TTP KO
mice to LPS, leading us to conclude that myeloid cell expression of
TTP under normal circumstances is a critical aspect of the body’s
defense mechanisms when confronted with Gram-negative bac-
terial infections. However, there are many other environmental
stimuli to TNF secretion, including both microbial products and
nonbiological agents, such as UV light and ionizing radiation. It
will be interesting to determine whether TTP plays a role in
protecting the body against the deleterious effects of TNF excess
in response to these other stimuli; the experimental models de-
scribed in this work should make it relatively straightforward to
test these ideas, in both cells and intact mice.
We thank Drs. Mark Hoenerhoff and Gordon Flake for assistance with
analysis of the histological slides, Dee Wenzel for animal husbandry sup-
port, Toni Ward for help with sample preparation, and the National Institute
of Environmental Health Sciences histology facility for tissue processing,
immunostaining, and photography. We also thank Drs. Donald N. Cook
and Michael B. Fessler for helpful comments on the manuscript.
The authors have no financial conflicts of interest.
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