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ipopolysaccharide signaling in the carotid chemoreceptor pathway ofats with sepsis syndrome
icardo Fernándeza,∗, Gino Nardoccia, Felipe Simona, Aldo Martina, Alvaro Becerraa,arolina Rodríguez-Tiradob, Kevin R. Maiseyb, Claudio Acuna-Castillob, Paula P. Cortesa
Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas y Facultad de Medicina, Universidad Andres Bello, 837 0134 Santiago, ChileDepartamento de Biologia, Facultad de Quimica y Biologia, Universidad de Santiago de Chile, 917 0022 Santiago, Chile
r t i c l e i n f o
rticle history:ccepted 22 December 2010
eywords:arotid bodyepsisPS
a b s t r a c t
In addition to their role in cardiorespiratory regulation, carotid body (CB) chemoreceptors serve as sensorsfor inflammatory status and as a protective factor during sepsis. However, lipopolysaccharide-inducedsepsis (LPS) reduces CB responsiveness to excitatory or depressant stimuli. We tested whether LPS exertsa direct effect on the carotid chemoreceptor pathway, the CB and its sensory ganglion. We determinedthat the rat CB and nodose–petrosal–jugular ganglion complex (NPJgc) express TLR4, TNF-� and its recep-tors (TNF-R1 and TNF-R2). LPS administration (15 mg/kg intraperitoneally) evoked MyD88-mechanism
LR4yD88-dependent
NF-�
pathway activation in CB and NPJgc, with NF-�B p65, p38 MAPK, and ERK activation. Consistently, LPSincreased TNF-� and TNF-R2. Double-labeling studies showed that the aforementioned pathway occursin TH-containing glomus cells and NPJgc neurons, components of the chemosensitive neural pathway.Thus, our results suggest that LPS acting directly through TLR4/MyD88-mechanism pathways increasesTNF-� and TNF-R2 expression in the carotid chemoreceptor pathway. These results show a novel affer-
l nernd th
ent pathway to the centrasepsis pathophysiology a
. Introduction
It is widely accepted that there is a bidirectional relationshipetween the central nervous system (CNS) and the immune sys-em. However, while brain-to-immune communications have beenroadly studied, the question of how immune information signalso the brain is still controversial. In recent years, increasing evi-ence has indicated that the vagus nerve may play an importantole in this regard (Goehler et al., 2000). Inflammatory media-ors released by immune cells could activate vagal paragangliand primary afferent neurons from their sensory ganglia (Hosoit al., 2005). In consequence, immunosensory inputs could initiateocal cardiorespiratory reflexes and carry information about thetate of inflammation. Systemically, subdiaphragmatic vagotomy
uppresses lipopolysaccharide (LPS)-induced sickness syndromereviewed by Watkins et al., 1995). At the molecular level, Wan et al.1994) demonstrate that subdiaphragmatic vagotomy completelylocked the hypothalamic and brain stem induction of c-Fos protein
∗ Corresponding author at: Laboratorio de Fisiologia, Departamento de Cienciasiologicas, Facultad de Ciencias Biologicas y Facultad de Medicina, Universidadndres Bello, Av. Republica 252, 837 0134 Santiago, Chile. Tel.: +56 2 661 5650;
evoked by the intraperitoneal (I.P.) administration of LPS; but hada minimal effect on c-Fos protein induction when LPS was admin-istered intravenously (I.V.) (Wan et al., 1994). Vagal afferences endprimarily within the nucleus tractus solitarii (NTS), and LPS-inducedc-Fos activation of NTS neurons also persists after cervical bilateralvagotomy (Hermann et al., 2001).
The systemic inflammatory response induced by LPS is dueto host cells stimulation (monocytes/macrophages, endothelial,and polymorphonuclear cells) to produce and release endoge-nous mediators, like reactive oxygen species and pro-inflammatorycytokines (tumor necrosis factor-� (TNF-�), interleukin-1 (IL-1),and IL-6) (Schletter et al., 1995). However, in spite of the expressionof IL-1 receptor in vagal glomus cells (GCs) (Goehler et al., 1997),IL-1� has no significant effects on the frequency of action potentialsrecorded from single fibers innervating isolated superfused rat GCsobtained from vagal nerve paraganglia (Mac Grory et al., 2010).Thus, this evidence suggests that immune chemosensory inputsand incoming neural signals could originate from other receptors,such as the carotid chemoreceptor pathway: the carotid body (CB)and its sensory ganglion. From an anatomical standpoint, the CB
is the largest paraganglion in the body. It is innervated essentiallyby sensory neurons which perikarya reside mainly in the petrosalganglion (Kalia and Davies, 1978), and the first synapse at the CNSfor afferent carotid sinus nerve (CN) fibers occurs in the NTS (Finleyand Katz, 1992).
Increasing evidence supports the idea that carotid chemore-eptors could play an important role in sensing and transmittingmmune signals to the CNS. The expression of type-I IL-1 recep-or (IL-1RI) (Wang et al., 2002; Shu et al., 2007), IL-6 receptorlpha chain (IL-6R�) (Wang et al., 2006), and type-1 tumor necro-is factor-� (TNF-�) receptor (TNF-R1) (Lam et al., 2008) in theCs of the rat CB; and TNF-� and TNF-R1 in the cat CB (Fernandezt al., 2008) suggest that carotid chemoreceptor function may benfluenced by inflammatory cytokines.
Recently, we demonstrated that LPS-induced tachypnea is pre-ented by bilateral section of the carotid and aortic nerves innesthetized cats, and that LPS enhances tonic CB chemosensoryctivity, but reduces its responsiveness to transient excitatory orepressant stimuli (Fernandez et al., 2008), as has been described
n newborn piglets (McDeigan et al., 2003) and rats (Ladinot al., 2007) subjected to endotoxin infusion. Since diminishedhemosensory activity observed in LPS-treated animals is notue to a decrease in functional tissue (Fernandez et al., 2008),he participation of systemic or locally produced factors is sug-ested. LPS administration increases cytokine plasma levels inany species. Thus, by using in vitro perfused and superfused CB
reparations, we demonstrate that the enhanced CB chemosensoryischarges recorded in responses to hypoxic stimulation are tran-iently diminished by TNF-� (Fernandez et al., 2008). In addition,L-1� application to carotid body GCs raises intracellular calcium,nd increases CN firing (Shu et al., 2007), and IL-6 increases intracel-ular calcium and induces catecholamine secretion from GCs (Fant al., 2009).
In this report we studied the expression of the LPS receptor andhe direct effect of LPS on the carotid chemoreceptor neural path-ay – CB and nodose–petrosal–jugular ganglion complex, NPJgc –
n a rat model of experimental endotoxemia. Here we demonstrateor the first time that (1) both carotid body GCs and chemosensoryeurons from NPJgc express LPS canonical receptor TLR4, MyD88,NF-�, TNF-R1 and TNF-R2; (2) LPS-induced TLR4 activation evokes�B� degradation, NF-�B translocation in the nuclei from CB andPJgc chemosensory cells, and both p38 MAPK activation and ERKctivation; and (3) LPS increases tyrosine hydroxylase, TNF-�, andNF-R2 expressions in the dopaminergic chemosensitive pathway.
. Methods
.1. Animals, cardiorespiratory recordings and tissue extraction
Experiments were performed on male Sprague–Dawley rats,eighing from 90 to 110 g. Experimental protocols were approved
y the Commission of Bioethics and Biosafety of the Universidadndres Bello.
Animals were anaesthetized with sodium pentobarbitone0 mg/kg I.P. (kindly provided by Dr. Patricio Zapata) and placed
n a supine position. The animals breathed spontaneously through-ut the experiment and surgical procedures. Body temperature,ssessed with a rectal thermistor probe, was maintained at about7.0 ± 0.1 ◦C, by placing a regulated heating pad under the rat. Ani-als were separated into two groups: a control group, treated withsaline solution (saline) and the experimental group, treated with5 mg/kg lipopolysaccharide (LPS, from Escherichia coli Serotype127:B8. L-3129 Sigma-Aldrich Corp, USA), both given I.P.
To confirm the effectiveness of LPS treatment – i.e., the induc-ion of severe sepsis – systolic blood pressure, instantaneous heart
requency, tidal volume, and instantaneous respiratory frequencyere continuously recorded before and for up to 2 h after saline-
r LPS-treatment, with a physiological recording acquisition sys-em PowerLab® 8/30 (AD Instruments, Castle Hill, Australia) (seeupplementary Table S1).
& Neurobiology 175 (2011) 336–348 337
To gain access to the carotid regions on both sides, aventral midline incision of the neck was performed. Carotidchemoreceptors, which include the carotid body (CB) and thenodose–petrosal–jugular ganglion complex (NPJgc), were excisedseparately and immediately transferred into cold-buffered Hanks’solution for 5 min, to allow complete blood cleaning. The main por-tion of the nodose ganglion was then excised at the point at whichthe IX and X cranial nerves were in close proximity. Carotid bod-ies and NPJgc were excised from different animals before salineor LPS treatment, and at different times (15, 30, 60, and 120 min)after injections (Supplementary Fig. S1). Due to the small size ofboth CBs and NPJgc, the organs obtained from 4 rats (i.e., 8 CBs or8 NPJgc separately) were pooled and stored in Trizol-reagent, lysisbuffer, or buffered 4% p-formaldehyde, depending on the follow-ing procedures (see below). At the end of the experiments, animalswere euthanized by an overdose of pentobarbitone.
2.2. Cell culture
Primary cultures of GCs and chemosensory neurons wereobtained by combined enzymatic and mechanical dissociationof CB (Nurse and Fearon, 2002) and NPJgc (Zhong and Nurse,1997), respectively. Briefly, CBs and NPJgc were excised fromuntreated pentobarbitone-anesthetized rats (N = 4) and kept at 4 ◦Cin modified Hanks’ balanced salt medium (HBS, Sigma–Aldrich)at pH 7.4, supplemented with 4 mM NaHCO3 and 5 mM HEPES(Sigma–Aldrich). Thereafter, tissues were mechanically dissoci-ated with forceps and triturated to yield cell suspensions, whichwere transferred to an enzymatic solution containing 0.1% colla-genase, 0.1% trypsin, 150 U/mL DNase I (all from Sigma–Aldrich)in HBS and incubated for 1 h at 37 ◦C. Following enzyme inactiva-tion, cell suspensions were plated in an 8-well Lab-TekTM II-CC2
Chamber Slide SystemTM (Nunc, Thermo Fisher Scientific, Inc.) andcultured at 37 ◦C in a humidified atmosphere of 95% air–5% CO2for 48 h in Ham’s F-12 nutrient mixture (Sigma–Aldrich) supple-mented with 10% horse serum (Gibco), 26 mM NaHCO3, 80 UI/LInsulin (ActrapidTM HM, Novo Nordisk A/S, Denmark), 0.6% glu-cose, 2 mM l-glutamine and 1% penicillin–streptomycin (Gibco).After 48 h in vitro culture period, cells were processed for RT-PCR,Western blot and immunocytochemistry, as described below.
2.3. Semiquantitative RT-PCR procedure
Total RNA from CB and NPJgc extracted from saline- and LPS-treated rats was isolated using Trizol reagent (Invitrogen, Carlsbad,CA) according to the manufacturer’s instructions. Quantificationand purity check of total RNA were performed spectrophotometri-cally and electrophoretically, respectively. After purification, RNAsamples were treated with DNAse-I (1 U/�L) (Invitrogen) to elim-inate genomic DNA. For in vitro cultured GCs and NPJgc neurons,100 �L Trizol reagent were directly applied into the culture wellsand RNA was extracted after three consecutive mixing (aspira-tion/dispensing) cycles.
Total RNA (2 �g) was reverse transcribed into cDNA usingMoloney Murine Leukemia Virus Reverse Transcriptase (M-MLVRT, 200 U/�L, Invitrogen). Reaction tubes were incubated at 42 ◦Cfor 50 min. At the end of the incubation period, the reaction wasstopped by heating at 90 ◦C for 5 min. PCR amplification was per-formed in a final reaction volume of 50 �L, containing 2 �L of thecDNA mixture diluted with the reaction buffer (10×) to a final con-centration of 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2,
200 �M dNTPs, 2.5 U Taq DNA polymerase (Life Technologies, Inc.),and 30 pmol of each primer.
The primer pairs used to amplify the coding regions of ratgenes for Toll-like receptor-4 (TLR4), myeloid differentiation factor88 (MyD88), cluster of differentiation molecule 14 (CD14), tumor
338 R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348
Table 1Oligonucleotide primers used in this study.
Genes Sequence Fragment size (bp) References
TLR4 Forward 5′-TTGAAGACAAGGCATGGCATGG-3′
Reverse 5′-TCTCCCAAGATCAACCGATG-3′508 Lehnardt et al. (2002)
MyD88 Forward 5′-AGTTGCTAGCCTTGTTAGACCGTGAGG-3′
Reverse 5′-AAACAACCACCACCATGCGACGACACC-3′399 Fujisawa et al. (2006)
CD14 Forward 5′-GATCTGTCTGACAACCCTGAGT-3′
Reverse 5′-GTGCTCCTGCCCAGTGAAAGA-3′267 Lehnardt et al. (2002)
TNF-� Forward 5′-TGGCCCAGACCCTCACACTC-3′
Reverse 5′-TCATACCAGGGCTTGAGCTCAG-3′361 Raina and Jeejeebhoy (2004)
TNF-R1 Forward 5′-ACCAAGTGCCACAAAGGAACC-3′
Reverse 5′-TACACACGGTGTTCTGTTTCTCC-3′322 Lung et al. (2001)
TNF-R2 Forward 5′-ATGAGAAATCCCAGGATGCAG-3′
Reverse 5′-ACAGACGTTCACGATGCAGGTG-3′254 Lung et al. (2001)
CD11b Forward 5′-ATGTGGACTCTGATGCCTCC-3′
Reverse 5′-TGTCTGAGCCTTCACAAACG-3′629 Dolman et al. (2005)
ecrosis factor-� (TNF-�), type-1 and type-2 TNF-� receptors (TNF-1 and TNF-R2, respectively), cluster of differentiation molecule1b (CD11b), cluster of differentiation molecule 163 (CD163), tyro-ine hydroxylase (TH), and 28S ribosomal RNA are shown in Table 1.n order to compare the results from different experiments, opti-
al cycle conditions for linear amplification were determined bysemiquantitative assay of the amplified products over a range of
ycles (ranging from 25 to 34). Products from 29 cycles were withinhe linear phase of the amplification curve and were thereforehosen for comparative analysis. Since hypoxia, the natural stimu-us for arterial chemoreceptors, modifies the expression of �-actin
RNA, each experiment included 28S amplification as an internalontrol (Zhong and Simons, 1999). For quantification, results wereormalized to 28S band intensity.
The tubes were placed in a Thermal Cycler (Mastercycler®,ppendorf AG, Germany) as follows: incubation at 94 ◦C for 4 mininitial melt); 29 cycles of the following sequential steps: 94 ◦C for0 s (melt); 55 ◦C for 45 s (anneal); 72 ◦C for 60 s (extend); and2 ◦C for 10 min (final extension). Total RNA obtained from lungas used as a positive control for TLR4, MyD88, CD14, TNF-�, TNF-1, and CD163; from adrenal gland, for TH; and, from lung excised
rom endotoxemic rats, for CD11b and TNF-R2. As negative controls,eactions were also carried out using samples without RNA or withDNA preparations prepared in the absence of reverse transcriptasenzyme. The amplified products were separated on a 2% agarose gel,hich was subsequently stained with ethidium bromide (Sigma)
nd photographed under UV illumination. Images were acquiredith a digital camera and intensity values were obtained with the
mageJ software (National Institutes of Health, Bethesda, MD).
.4. Western blot
Excised CB and NPJgc were homogenized using a reusabletainless 0.5 mL pellet pestle (Thomas Scientific) in a minimummount of ice-cold lysis buffer [50 mM Hepes, 150 mM NaCl, 1 mMGTA, 10% glycerol, 2 mM MgCl2, 1% Triton X-100, 0.1% SDS,
.1% IGEPAL CA-630 and 0.1% Protease Inhibitor Cocktail (P8340,igma–Aldrich)] and centrifuged (11,000 × g for 20 min at 4 ◦C). Tis-ues used as positive controls, from either saline- or LPS-treatedats, were homogenized with a speed-regulated tissue homog-nizer. The supernatants were mixed with loading buffer (final
concentration: 100 mM Tris–HCl, pH 6.8, 4% SDS, 10% glycerol, 0.2%bromophenol blue). To obtain total proteins from in vitro culturedGCs and NPJgc neurons, 50 �L loading buffer (2×) were directlyapplied into the culture wells and, after three consecutive mixingcycles, protein samples were frozen until use.
Proteins were resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred tonitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in TBS-Tween (100 mM Tris–HCl, pH 7.5, 0.9% (w/v)NaCl and 0.1% Tween 20) at room temperature for 1 h. Mem-branes were then incubated with primary antibodies against TLR4,MyD88, CD14, nuclear factor (NF)-�B p65, inhibitory (I)�B�, p38mitogen-activated protein kinase (p38 MAPK), phosphorylated (p)-p38 MAPK, extracellular signal-regulated protein kinase (ERK),phosphorylated (p)-ERK, TNF-�, TNF-R1, TNF-R2, CD11b, CD163,and TH (Table 2) overnight at 4 ◦C. Then, membranes were washedand incubated with their respective peroxidase-conjugated sec-ondary antibodies by 1 h at room temperature. After washing,peroxidase activity was detected through enhanced chemilumines-cence (Bio-Rad, CA, USA). After stripping, membranes were probedfor �-actin as loading control. Immunoreactive bands were scannedand intensity values were obtained by densitometry of individualbands, normalized against �-actin. Molecular weights of proteinswere estimated by using pre-stained Kaleidoscope marker (Bio-Rad).
2.5. Immunofluorescence and digital confocal microscopyanalysis
Excised CBs and NPJgc were fixed in cold 4% (w/v) p-formaldehyde in phosphate buffered saline (PBS, pH 7.4) for 1 hat 4 ◦C, before sequential transfer to 10% (w/v) sucrose in PBS for1 h at 4 ◦C, and to 30% (w/v) sucrose in PBS overnight at 4 ◦C.In vitro cultured GCs and NPJgc neurons were fixed and perme-abilized for TLR4, TH and neuron-specific nuclear protein NeuronalNuclei (NeuN) detection. Tissue samples were embedded in freez-
ing medium (Tissue-Tek® OCT Compound, Electron MicroscopySciences, Hatfield, PA) at −20 ◦C. Thereafter, cryostat sections of12 �m thickness were obtained and mounted on gelatin-coatedslides, and stored at 4 ◦C until use (no more than one week). TLR4,MyD88, NF-�B p65, TNF-�, TNF-R1, TNF-R2, and TH proteins were
R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348 339
Table 2Source and dilution of antibodies used in this study.
ifferentiation molecule 163; Ig, immunolglobulin; HRP, Horseradish peroxidase; Bl.u.: not used. (1) Santa Cruz Biotechnology, Inc. (2) Cell Signaling Technology, Inc. (36) BiosourceTM, Life Technologies.
dentified in CB and NPJgc tissues. Briefly, slices were rehydratedith PBS for 1 h at room temperature. Then, formaldehyde-induceduorescence was quenched by incubation with 50 mM NH4Cl inBS for 20 min at 4 ◦C. After rinsing, slices were blocked and per-eabilized in PBS containing 1% (w/v) bovine serum albumin (BSA,
igma–Aldrich), 5% sucrose, and 1% Triton X-100 by 2 h at roomemperature. After washing, sections were incubated overnight at◦C with primary antibodies (Table 2). After incubations, the sec-
ions were washed and incubated for 1 h at 37 ◦C with either Alexaluor® 488- or 568-conjugated secondary antibodies.
To identify CB and NPJgc chemosensitive cells, double-labelingf TH-containing type-1 (glomus) cell clusters (Eyzaguirre andapata, 1984) and nodose–petrosal neurons (Katz et al., 1983) wasetected by overnight incubation at 4 ◦C with rabbit anti-rat TH.s an exception mouse anti-rat TH was used for NF-�B p65-THouble-labeling studies (Table 2). Sections were then washed and
ncubated with Alexa Fluor®-conjugated secondary antibodies. Allhe antibodies were diluted in PBS containing 1% BSA and 0.3%riton X-100. For NF-�B p65 translocation studies, nuclei weredditionally stained by incubation with Hoechst 33342 (Molecularrobes, Invitrogen).
Finally, slides were washed and permanently mounted in 10%v/v) 1,4-diazobicyclo-[2.2.2]octane (DABCO, Sigma–Aldrich) and0% (v/v) glycerol. Staining specificity was determined by the incu-
ation of sections in the absence of the primary antibody and/ory pre-adsorption with specific blocking peptides when availableTable 2, Supplementary Fig. S2) (Fritschy, 2008). In some slides,uclei were stained with TO-PRO®-3 iodide (T3605 Molecularrobes, Invitrogen). Samples were examined using laser scanning
locking peptide; IHC, immunohistochemistry; WB, Western blot; n.a.: not available;micon International, Inc. (4) Sigma–Aldrich, Inc. (5) InvitrogenTM, Life Technologies.
confocal microscopy on either an Axiovert 100 Microscope (CarlZeiss, Inc.) or a Fluoview FV1000 (Olympus).
2.6. Data analysis
Data were expressed as Means ± Standard Deviation (SD) of theMean. Statistical differences were assessed by Mann–Whitney testor by non-parametric analysis of variance (ANOVA, Kruskal–Wallistest) followed by Dunns’ post-test, as indicated in legends forthe figures, and considered significant when p < 0.05. For NF-�B p65 translocation studies, samples obtained from saline- andLPS-treated rats were analyzed with ImageJ software (NationalInstitutes of Health, Bethesda, MD). Briefly, nuclei fluorescence wasnormalized against the area (pixels/�m2) and background fluores-cence was subtracted.
3. Results
3.1. TLR4 expression in the carotid chemoreceptor pathway
To investigate whether the LPS canonical receptor mechanismcan mediate endotoxin-induced CB inflammation and diminishedcarotid chemosensory activity, the expression of TLR4, and itsintracellular signaling mediators, were investigated in CB and
NPJgc. Since LPS increases TLR4 expression in many rat tissues, thechanges in TLR4 expression in the carotid chemoreceptor pathwayfrom endotoxemic rats were also studied. By means of RT-PCR, anexpected fragment corresponding to TLR4 mRNA in both CB andNPJgc was amplified (Fig. 1A, upper panels). In addition, a sin-
340 R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348
Fig. 1. Expression of LPS receptor and accessory molecules in the carotid chemoreceptor neural pathway. (A) Toll-like receptor 4 (TLR4) mRNA and protein were detectedin the CB and NPJgc obtained 2 h after saline (−) or 15 mg/kg LPS (+) I.P. administration. Upper panels: mRNA expression of TLR4 (arrow) and 28S (arrow head). Lower panels:T and pro 88 (MN ntrols
glp(otebu
aBCpoaeicbu
3f
acwre
LR4 (arrow) and �-actin (arrow head) protein immunodetection. (B) TLR4 mRNAf cluster of differentiation molecule 14 (CD14) and myeloid differentiation factorPJgc. Images are representative of at least three separate experiments. Positive co
le band corresponding to TLR4 protein was also found (Fig. 1A,ower panels). Two hours after LPS challenge, neither mRNA norrotein levels were significantly modified in both CB and NPJgcFig. 1A). Densitometric analysis for N = 3–4 independent mRNAr protein extracts, normalized to either 28S or �-actin, respec-ively, confirms that LPS failed to evoke significant changes in TLR4xpression (p > 0.05, Mann–Whitney test, not shown). Additionally,oth in vitro cultured CB and NPJgc express the TLR4 mRNA (Fig. 1B,pper panel) and protein (Fig. 1B, lower panel).
Lipopolysaccharide-induced TLR4 activation requires theccessory molecule CD14 (Akashi-Takamura and Miyake, 2008).y means of RT-PCR, an expected fragment corresponding toD14 mRNA in both CB and NPJgc was amplified (Fig. 1C, middleanel). CD14 protein was also found (Fig. 1D, lower panel). More-ver, acting through TLR4, LPS activates both MyD88-dependentnd MyD88-independent pathways (Lu et al., 2008). Thus, thexpression of MyD88 in CB and NPJgc from saline-treated rats wasnvestigated. In both tissues, RT-PCR gives an expected fragmentorresponding to MyD88 mRNA (Fig. 1C, upper panel). Westernlot analysis showed that MyD88 protein is also expressed (Fig. 1D,pper panel).
.2. LPS signaling components are found in chemosensory cellsrom the carotid chemoreceptor pathway
To identify which cells from CB and NPJgc express TLR4 and the
ccessory molecule MyD88, double-labeling immunohistochemi-al experiments were carried out. Positive TLR4-immunoreactivityas located in CB lobules (Fig. 2A, left) and NPJgc neurons (Fig. 2A,
ight), which were also positive for TH, confirming LPS receptorxpression in chemosensitive GCs and neurons from either nodose
otein expression in GCs and NPJgc neurons cultured in vitro. (C) mRNA expressionyD88) in the CB and NPJgc. (D) CD14 and MyD88 protein expression in the CB andare shown for each experiment.
or petrosal ganglia. Double-labeling studies from in vitro culturedchemoreceptors confirm that TLR4 expression is present in carotidbody GCs, which were positive for TH, and in NPJgc neurons, whichwere are also positive for the neuronal marker NeuN (Fig. 2B).In addition, positive MyD88-immunoreactivity was located in CBlobules (Fig. 2C, left) and NPJgc neurons (Fig. 2C, right), whichwere also positive for TH. Altogether, these results confirm theexpression of LPS-canonical receptorTLR4 and MyD88-dependentmechanism in the carotid chemoreceptor neural pathway, i.e.,chemosensitive GCs and neurons from either nodose or petrosalganglia.
3.3. LPS induces I�B˛ degradation and NF-�B translocation intothe nucleus from chemosensitive cells of the carotidchemoreceptor pathway
Classically, LPS binding to TLR4 promotes I�B� degradation andsubsequent NF-�B translocation into the nucleus (Liu and Malik,2006) through a Myd88-dependent mechanism. Taking these factsinto consideration, I�B� degradation and NF-�B p65 translocationwere studied. Western blot analysis reveals that the I.P. admin-istration of LPS significantly reduced cytosolic I�B� in both CB(Fig. 3A) and NPJgc (Fig. 3B), within 15 min after LPS administra-tion, and remains low for 30 min post-LPS administration. Theseresults were similar to those obtained in liver tissue used as positivecontrol (Fig. 3C). Densitometric analysis for N = 3 independent pro-
tein extracts, normalized to �-actin, confirming that LPS decreasedcytosolic I�B� (p < 0.05, Kruskal–Wallis ANOVA, followed by Dunns’multiple comparison test).
Cytosolic NF-�B p65 detection decreased in both CB (Fig. 3D) andNPJgc (Fig. 3E) obtained from LPS-treated rats, as with a positive
R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348 341
Fig. 2. Immunolocalizations in the carotid chemoreceptor neural pathway. (A) Localization of TLR4 in histological sections from CB (left) and NPJgc (right) obtained fromsaline treated rats. Chemosensory GCs and NPJgc neurons were identified by their TH positive immunoreactivity. (B) TLR4 expression in GCs and NPJgc neurons culturedin vitro. TH and NeuN immunoreactivity were used for GCs and NPJgc neuron detection, respectively. (C) MyD88 localization in the CB and NPJgc obtained from saline treatedrats. GC clusters and chemosensory neurons were identified by TH co-localization. (D) Localization of TNF-� in histological sections from CB and NPJgc obtained 2 h aftersaline (upper panels) or LPS (lower panels) treatments. Chemosensory cells were identified by their TH immunoreactivity. After LPS, note a stronger TNF-� immunoreactivity inb tion op eactivb repres
lddf
oth CB and NPJgc. As well, observe the increased TH immunoreactivity. (E) Localizaanels) or LPS (lower panels) treatments. In CB (left), note a slight TNF-R2 immunoroth CB and NPJgc. As well, observe the increased TH immunoreactivity. Images are
iver control (Fig. 3F). Densitometric analysis for N = 3 indepen-ent protein extracts, normalized to �-actin, confirms that LPSecreased cytosolic NF-�B p65 (p < 0.05, Kruskal–Wallis ANOVA,ollowed by Dunns’ multiple comparison test). In order to corre-
f TNF-R2 in histological sections from CB and NPJgc obtained 2 h after saline (upperity surrounding CG clusters. After LPS, note a stronger TNF-R2 immunoreactivity inentative of at least three separate experiments. Scale bars are shown in the figures.
late the cytosolic diminution with the NF-�B p65 nuclear increase,immunofluorescence experiments were carried out in the histo-logical sections obtained from CBs and NPJgc excised from rats,60 min after saline or LPS treatment (Fig. 3G and H). In saline-
342 R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348
Fig. 3. MyD88-dependent mechanism activation in the carotid chemoreceptor neural pathway. Representative images from at least three separate experiments (upperpanels) showing the time course of I�B� degradation (arrow) in CB (A), NPJgc (B), and liver (C), and NF-�B p65 cytosolic fraction decrement (arrow), in CB (D), NPJgc (E), andliver (F), at control (t = 0), 15, and 30 min after LPS treatment. Arrowheads indicate the expression of �-actin used as loading control. Lower panels: densitometric analysis forN = 3 independent experiments, normalized to �-actin, and shown relative to 0 time. Values are Means ± SD. Statistical differences from saline-treated rats were assessed byKruskal–Wallis ANOVA, followed by Dunn’s multiple comparison test, *p < 0.05. (G and H) Immunoreactivity for NF-�B p65 in histological sections from CB (G) and NPJgc (H)o t threef ronst re Met
ti(Nblia
btained 60 min after saline or LPS treatments. Images are representative of at leasor NF-�B p65 nuclear immunoreactivity from both carotid body GCs and NPJgc neuo the analyzed area. The number of nuclei analyzed is shown in each bar. Values aest, *p < 0.05.
reated rats, an almost undetectable positive reaction was observedn the nuclei of both CB (Fig. 3G, left upper panel) and NPJgcFig. 3H, left upper panel). As expected, LPS administration evokes
F-�B p65 translocation into the nuclei of TH-positive carotidody GCs (Fig. 3G, left lower panel) and NPJgc neurons (Fig. 3H,
eft lower panel). Densitometric analysis showed a significantncrease in nuclear mark (p < 0.05, Mann–Whitney test) (Fig. 3Gnd H).
separate experiments. Scale bars are shown in the figures. Densitometric analysisobtained 60 min after either saline or 15 mg/kg LPS I.P. administration, normalizedans ± SD. Statistical differences were assessed by non-parametric Mann–Whitney
3.4. LPS induces p38 MAPK and ERK phosphorylation in thecarotid chemoreceptor pathway
Acting through a MyD88-dependent mechanism, LPS alsoinduces p38 MAPK and ERK activation (Lu et al., 2008). Thus, LPS-induced p38 MAPK and ERK phosphorylation (p-p38 and p-ERK,respectively) in the carotid chemoreceptor pathway was stud-ied. In CBs and NPJgc, both kinases were found (Fig. 4A–C). After
R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348 343
Fig. 4. Activation of ERK and p38 MAPK and TNF-� up-regulation in the carotid chemoreceptor pathway. (A–C) Representative images for at least three separate experimentsshowing the changes in CB phosphorylated fraction of ERK (p-ERK) (A), NPJgc p-p38 MAPK (B), and NPJgc p-ERK (C) at different times after 15 mg/kg LPS I.P. challenge.Lower panels: densitometric analysis for N = 3–4 independent protein extracts, normalized to �-actin and shown relative to control (t = 0). Values are Means ± SD. Statisticaldifferences from t = 0 were assessed by non-parametric Kruskal–Wallis test, followed by Dunns’ multiple comparison test, *p < 0.05. (D and E) Representative images from atleast three-separate RT-PCR (D) or Western blot (E) experiments showing the expression of TNF-� mRNA and protein (arrows) in the CB (left) and NPJgc (right) obtained 2 ha as usem headss
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fter saline (−) or LPS (+) I.P. administration. Lung tissue from saline-treated rats wRNA or protein extracts, normalized to either 28S or �-actin respectively (arrow
aline-treated rats were assessed by non-parametric Mann–Whitney test, *p < 0.05.
PS administration, p-p38 MAPK in the CB was not detected (nothown), but a marked increase in p-ERK fraction was found at0 min post LPS administration (Fig. 4A). In the NPJgc, LPS admin-
stration increased both p-ERK (Fig. 4C) and p-p38 MAPK (Fig. 4B),t 15 and 30 min, respectively. Densitometric analysis for N = 3–4ndependent protein extracts, normalized to �-actin, confirmshat LPS increased p-p38 MAPK/p38 MAPK and p-ERK/ERK ratiop < 0.05, Kruskal–Wallis ANOVA, followed by Dunns’ multiple com-arison test).
.5. TNF-˛ expression and upregulation induced by LPS in thearotid chemoreceptor pathway
In both CB and NPJgc, TNF-� mRNA (Fig. 4D) and protein wereound (Fig. 4E), confirming our previous results (Fernandez et al.,008). Given that LPS typically increases TNF-� expression, we
nvestigated whether LPS treatment modifies TNF-� expressionn the carotid chemoreceptor pathway. Two hours after LPS chal-enge, both mRNA and protein levels increased dramatically in theB and NPJgc (Fig. 4D and E). Densitometric analysis for N = 3–4
ndependent mRNA or protein extracts, normalized to either 28S
r �-actin, respectively, confirms that LPS increases TNF-� expres-ion (p < 0.05, Mann–Whitney test). The localization of TNF-�rotein in CB and NPJgc is also shown in Fig. 2D. Positive TNF-�-
mmunoreactivity was localized in CB lobules and in NPJgc neurons,hich were also positive for TH, confirming expression of TNF-�
d as positive control. Lower panels: densitometric analysis for N = 3–4 independent), and shown relative to saline. Values are Means ± SD. Statistical differences from
in chemosensitive cells (Fig. 2D, upper panels). As expected, LPSadministration increased TNF-� immunoreactivity. Two hours afterendotoxin treatment, both CB and NPJgc showed a stronger posi-tive immunoreactivity than saline-treated samples. In both CB andNPJgc, intense TNF-� staining was observed in TH-positive cells,confirming that LPS-induced TNF-� expression increases in GCs andchemosensory neurons (Fig. 2D, lower panels).
3.6. Expression of TNF-˛ receptors in the carotid chemoreceptorpathway
It is known that TNF-� exerts its biological effects through inter-action with TNF-R1 and TNF-R2. Therefore, the expression of bothTNF-� receptors in the carotid chemosensory pathway was inves-tigated. TNF-R1 mRNA and protein were detected in both CB andNPJgc. Two hours after LPS challenge, neither mRNA nor proteinlevels were significantly modified in CB or NPJgc (SupplementaryFig. S3A). Densitometric analysis for N = 3–4 independent mRNAor protein extracts, normalized to either 28S or �-actin, respec-tively, confirms that LPS failed to evoke significant changes inTNF-R1 expression (p > 0.05, Mann–Whitney test. Not shown). It
must be noted that positive TNF-R1 immunostaining was locatedin CB lobules and NPJgc neurons, which were also positive for TH,confirming the expression of TNF-R1 in chemosensitive GCs andneurons from either nodose or petrosal ganglia (Supplementary Fig.S3B).
3 ology & Neurobiology 175 (2011) 336–348
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Fig. 5. TNF-R2 and TH up-regulation in the carotid chemoreceptor pathway. (A andB) Representative images from at least three separate RT-PCR (A) or Western blot(B) experiments showing the expression of TNF-R2 mRNA and protein (arrows)in the CB (left) and NPJgc (right) obtained 2 h after saline (−) or 15 mg/kg LPS (+)I.P. administration. Lung tissue from LPS-treated rats was used as positive control.Lower panels: quantitative densitometric analysis for N = 3–4 independent mRNAor protein extracts, values are Means ± SD, normalized to either 28S or �-actinrespectively (arrowheads), and shown relative to saline. Statistical differences fromsaline-treated rats were assessed by non-parametric Mann-Whitney test, *p < 0.05.(C) Changes in TH mRNA (left) and protein (right) expression in the CB obtained 2 h
44 R. Fernández et al. / Respiratory Physi
Since bacterial LPS enhances not only TNF-� synthesis andelease, but also the expression of TNF-R2 (Nadeau and Rivest,999), the effect of LPS treatment on TNF-R2 expression in theB-NPJgc pathway was assessed. By means of RT-PCR, an expected
ragment corresponding to TNF-R2 mRNA was found in both CB andPJgc (Fig. 5A). In agreement with the above-mentioned result, a
ingle band, corresponding to the soluble isoform of TNF-R2 (Lainezt al., 2004), was found (Fig. 5B). Two hours after LPS challenge,oth TNF-R2 mRNA and protein increased significantly in CB andPJgc (Fig. 5A and B). Densitometric analysis for N = 3–4 inde-endent mRNA or protein extracts, normalized to either 28S or-actin, respectively, confirms that LPS increased TNF-R2 expres-ion (p < 0.05, Mann–Whitney test).
The localization of TNF-R2 protein in CB and NPJgc is also shownn Fig. 2E. A slight positive TNF-R2-immunoreactivity was local-zed surrounding carotid body GC clusters. GCs were identifiedy their positive TH-immunoreactivity (Fig. 2E, left upper pan-ls). Therefore, and as we reported before (Fernandez et al., 2008),NF-R2 did not co-localize with GCs. On the contrary, positive TNF-2-immunoreactivity was localized near the cell limit of NPJgceurons (Fig. 2E, right upper panels), which were also positive forH, confirming TNF-R2 expression in chemosensitive neurons. Asxpected, LPS administration increased TNF-R2 immunoreactivity.n fact, when histological sections were processed for TNF-R2, anntense and ubiquitous TNF-R2 staining surrounding CB lobules
as found (Fig. 2E, left lower panels). Additionally, NPJgc neu-ons from LPS-treated rats showed a more intense positive TNF-R2mmunoreactivity than observed in NPJgc neurons from saline-reated (Fig. 2E, right lower panels).
Although post-extraction treatment efficiently cleaned remnantlood from microdissected CB and NPJgc (see Methods), it wastill possible that local immune cells – i.e., resident macrophagesDvorakova et al., 2000) or LPS-induced infiltrating polymorphonu-lear cells (PMNCs) (Fernandez et al., 2008) – were responsible, ateast in part, for the results reported here. Therefore, we studiedhe expression of CD163, a protein that is exclusively expressed onesident macrophages (Polfliet et al., 2006), and CD11b, an immuneell marker, which is expressed in many immune cell types, but thexpression of which is strongly associated with either microgliactivation in the CNS (Goehler et al., 2006), or PMNC activation inungs (Kermarrec et al., 1998; Bless et al., 1999).
In spite of CD163 mRNA expression in two NPJgc samples, bothB and NPJgc were negative for CD163 protein (Supplementaryig. S4A). In addition, samples obtained from both saline- or LPS-reated rats, were essentially negative for CD11b mRNA and proteinxpression (Supplementary Fig S4B and C). These observations indi-ate that under our experimental conditions, results here reportedre not attributable to resident macrophages or to infiltratingMNCs.
.7. LPS induces TH upregulation in the carotid chemoreceptorathway
Tyrosine hydroxylase (TH) is the rate-limiting enzyme for cate-holamine synthesis. Carotid body chemosensory GCs express THEyzaguirre and Zapata, 1984), and its expression is up-regulatedy I.P. administration of IL-1� (Zhang et al., 2007). Thus, TH expres-ion was assessed in the CB from endotoxemic rats. In CBs obtainedrom saline-treated rats, TH mRNA and protein were found (Fig. 5C).wo hours after LPS-administration, both mRNA and protein levelsncreased (Fig. 5C). Densitometric analysis for N = 3–5 independent
RNA or protein extracts, normalized to either 28S or �-actin,espectively, confirms that LPS treatment increases TH expres-ion (p < 0.05, Mann–Whitney test). TH localization was confinedo carotid body GCs and NPJgc neurons (Fig. 2). LPS-treatment alsoncreased TH immunoreactivity (Fig. 2D and E).
after saline (−) or LPS (+). Adrenal tissue from saline-treated rats was used as posi-tive control. Lower panels: densitometric analysis for N = 3–5 independent extracts,normalized to either 28S or �-actin respectively, and shown relative to saline. Val-ues are Means ± SD. Statistical differences from saline-treated rats were assessed bynon-parametric Mann–Whitney test, *p < 0.05.
R. Fernández et al. / Respiratory Physiology & Neurobiology 175 (2011) 336–348 345
Fig. 6. Proposed model for LPS signaling in the carotid chemoreceptor pathway from endotoxemic rats. Carotid body GCs constitutively express TLR4, MyD88, TNF-�,and TNF-R1. Carotid (sinus) nerve terminals (i.e., NPJgc neurons) also express TNF-R2. Plasma LPS, interacting with LPS-binding protein (LBP) and with either soluble ormembrane-anchored CD14 (sCD14 or mCD14), is transferred to TLR4/MD2 receptor complex. LPS interaction with TLR4, expressed in carotid chemosensory cells activatesthe MyD88-dependent mechanism, which induces a fast I�B� degradation and subsequent NF-�B translocation into the nucleus of carotid body GCs and NPJgc neurons.A n ande Jgc net S increr
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dditionally, LPS evokes p38 MAPK activation in GCs, and both p38 MAPK activatioxpression and neuronal TNF-R2. TNF-� could interact with TNF-R1 from GCs or NPractus solitarii (NTS). TNF-� could also be released directly in the NTS. Systemic LPeceptors. EC, endothelial cell; CN, carotid nerve.
. Discussion
This is the first study to demonstrate the expression of TLR4 inarotid body GCs and in NPJgc chemosensory neurons. Even more,his receptor appears to be functional, since upon systemic inflam-
atory challenge induced by the I.P. administration of LPS to rats,ndotoxin induces NF-�B p65, p38 MAPK, and ERK activation. Con-istent with the above-mentioned results, LPS treatment increasesoth mRNA and protein levels of TNF-� and type-2 TNF-� receptorsee Fig. 6 for details).
Sepsis is currently viewed as a complex dysregulation of themmune system, leading to uncontrolled production of soluble
ediators, which affect multiple organs by altering endothelial,pithelial, and immune cell responses that lead to irreversiblearenchymatous organ damage. Systemic inflammation asso-iated with sepsis involves many pathological processes, likenflammation per se, tachypnea, fever, leukopenia, hemodynamicbnormalities and hypotension, tachycardia, multiple organ dys-unction (MOD), and death (Riedemann et al., 2003). In agreementith this, LPS administration decreased systolic blood pressure, and
ncreased heart rate, respiratory frequency, and total ventilation.hus, our animal model of systemic inflammation complies withhe accepted criteria for the diagnosis of severe sepsis in humansBone et al., 1992; Levy et al., 2003).
The nervous system – acting through the autonomic nervousystem – coordinates the fine-tuning of cardiorespiratory interplay,o maintain an appropriate oxygen delivery in order to preventepsis-induced MOD. Autonomic balance is maintained by sev-ral reflex arcs, like arterial baroreflexes (Kirchheim, 1976), centralhemoreflexes, peripheral chemoreflexes, and pulmonary stretcheflexes (Liljestrand, 1958), which represent the major componentsf blood pressure and breathing regulation. The interactions amonghese reflexes are of special clinical interest, since the overactivity
f a single reflex, occurring pathophysiologically in several dis-rders, can lead to the suppression of opposite reflex responsesr vice versa (Schmidt et al., 2001). In this way, the first line fornjury recognition could be related to peripheral chemoreceptorensory neurons, the first CNS synapse of which occurs in the
ERK activation in NPJgc. NF-�B, p38 MAPK, and ERK co-activation increases TNF-�urons or with neuronal TNF-R2 and modify CB chemosensory inputs to the nucleusase plasma TNF-�, which in fact could interact with both GCs and neuronal TNF-�
NTS (Finley and Katz, 1992), a part of the dorsal vagal complex(DVC) (Berthoud and Neuhuber, 2000). Thus, inflammation-derivedsensory input originated systemically or locally – from peripheralchemoreceptors – can be differentially processed in the medulla,either modifying cardiorespiratory chemoreflexes, endocrine func-tion, or sensing the state of inflammation. Increasing lines ofevidence show that arterial chemoreceptors may play an importantrole during sepsis. For instance, endotoxemic cats show significantmodifications in chemoreceptor activity, such as increased basalchemosensory activity and decreased ventilatory chemoreflexesand ventilatory chemosensory drive. As well, increased respiratoryfrequency is prevented by bilateral section of the carotid and aorticnerves (Fernandez et al., 2008). Thus, CB chemoreceptors may pro-vide an afferent pathway for the elicitation of sepsis signs inducedby LPS.
Lipopolysaccharide could directly activate the CNS throughperipheral visceral sensory ganglia activation (Hosoi et al., 2005).But we support the idea that carotid chemoreceptors participate inCNS activation describing CB histological manifestations of acuteinflammation and functional changes in response to local or sys-temic endotoxin administration (Fernandez et al., 2008) and, in thiswork, have shown that the carotid chemoreceptor pathway – i.e.,the CB and its sensory ganglion (NPJgc in rats) – express functionalLPS canonical receptor, TLR4.
Besides TLR4, LPS recognition requires accessory molecules, likeCD14 and MD-2 (Akashi-Takamura and Miyake, 2008). We foundthat CB and NPJgc express CD14. However, we did not further inves-tigate CD14 localization in GCs or NPJgc neurons because in ouranimal model of sepsis, the presence of membrane-anchored CD14in the chemoreceptors could be less important since during sepsis,both monocytes (Durieux et al., 1994) and hepatocytes (Liu et al.,1998) secrete soluble CD14 to bloodstream, which in fact reachescarotid chemoreceptors because of their extensive vascularization
(Verna, 1979).
Since the I.P. administration of LPS stimulates vagal primaryafferent fibers, which in turn activate CNS neurons responsible forsystemic manifestations of sepsis (Mascarucci et al., 1998; Borsodyand Weiss, 2005), and the number of neurons expressing c-Fos
3 ology
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46 R. Fernández et al. / Respiratory Physi
ithin the DVC, after peripheral administration of LPS, correlatesith plasma levels of TNF-� (Hermann et al., 2001), it is possi-
le to suggest that TNF-� stimulates c-Fos activation of neuronsn the NTS. However, this cytokine has no significant effects on therequency of action potentials recorded from single fibers from iso-ated superfused rat GCs obtained from vagal nerve paragangliaMac Grory et al., 2010). Thus, the carotid chemoreceptor pathwayeems as a good candidate for mediating these responses.
Lipopolysaccharide challenge activates both MyD88-dependentnd MyD88-independent mechanisms; the former mainly for thenduction of pro-inflammatory cytokines and the latter for thenduction of Type I interferons (Lu et al., 2008). Results reportedere show that carotid body GCs and chemosensory neurons fromPJgc express functional MyD88-dependent pathway downstream
o the TLR4 receptor, which in fact results in the activation ofnducible transcription factors, such as NF-�B. Similarly, LPS actinghrough TLR4 and MyD88-dependent pathway activates mitogen-ctivated protein kinases (MAPKs), such as those for ERK 1 and 2ERK1/2), and p38 MAPK (Chang and Karin, 2001). However, this
echanism appears to be different in carotid body GCs and NPJgceurons. With endotoxin challenge, both CB and NPJgc increase thehosphorylated fraction of ERK. But, LPS increases the phosphory-
ated fraction of p38 MAPK just in NPJgc. Activation of ERK1/2 haseen linked to cell survival, whereas that of p38 MAPK to apoptosisSubramaniam and Unsicker, 2006). Interestingly, LPS treatmentnduced CB ERK phosphorylation, in the absence of p38 MAPK acti-ation. Then, this response could be a protective mechanism tovoid chemoreceptor dysfunction. These results concur with ourrevious work in which we were unable to detect apoptosis in theBs obtained from endotoxemic cats (Fernandez et al., 2008).
The activation of ERK in NPJgc neurons is quite different. Arowing number of studies have suggested a death-promoting roleor ERK1/2 in both in vitro and in situ models of neuronal deathSubramaniam and Unsicker, 2010). However, neuronal survivalr death strongly depends on the time of activation. In fact, sus-ained ERK1/2 activation promotes cell survival. Results obtainedere show significant ERK activation at 15 and 30 min post-LPSdministration, but we did not test other times (shorter or longer),n order to distinguish between transient or sustained activa-ion. Independent of the mechanisms involved, the co-activationf NF-�B, ERK1/2, and p38 MAPK results in the release of pro-nflammatory mediators, like IL-1�, TNF-� and IL-6, by astrocytesnd microglia (Kinsner et al., 2005). Thus, it is possible that, inesponse to systemic LPS, chemosensory NPJgc neurons producero-inflammatory cytokines, like TNF-� (this report).
Chemosensitive cells from the CB and NPJgc significantlyncreased their basal levels of TNF-� during sepsis. Thus, theeleased cytokine could act peripherally, in an autocrine/paracrineanner, directly on GCs or petrosal sensory nerves at the CB,
hrough TNF-R1 and/or TNF-R2, the latter also increased duringepsis. In fact, increasing doses of this cytokine decrease hypoxichemosensory activity (Fernandez et al., 2008), possibly by induc-ng GCs to release inhibitory transmitters, such as dopamine (DA),he predominant catecholamine synthesized in GCs (Zapata, 1975;turriaga et al., 1994). So, TNF-� could also act centrally, within theeural circuitry of the medullary DVC, to evoke complex autonomic,euroendocrine, metabolic, and behavioral responses to infectionnd inflammation.
It must be noted that plasma LPS could initially interactith endothelial cells, which in fact produce and secrete pro-
nflammatory cytokines in response to LPS. But in vitro cultured
uman umbilical vein endothelial cells (HUVECs) respond to LPSy increasing TNF-� mRNA expression 1 h after exposure, withoutetectable levels of TNF-� protein in the culture medium and cell
ysate for up to 8 h (Imaizumi et al., 2000). Thus, although the CBsre highly vascularized organs, endothelial cell TNF-� expression
& Neurobiology 175 (2011) 336–348
and secretion is later (Neuhaus et al., 2000). Similar results wereobtained by us, where LPS failed to evoke significant changes incytokine mRNA expression in HUVECs for up to 1 h (Simon andFernandez, 2009). Moreover, co-localization studies with TH, anaccepted marker of both carotid body GCs (Eyzaguirre and Zapata,1984) and NPJgc chemosensory neurons (Katz et al., 1983), stronglysupport the idea that in spite of increased plasma levels of TNF-�induced by systemic LPS, the increased TNF-� levels in CB and NPJgcare due mainly to chemosensory cell activation.
Type-1 TNF-� receptor is constitutively expressed in most tis-sues and appears to be the key mediator of TNF-� signaling,whereas expression of TNF-R2 is highly regulated and is typi-cally found in cells of the immune system (Wajant et al., 2003),but chemosensory neurons from NPJgc express TNF-R2, and LPSincreases its expression. TNF-R2 also increased in the rat brain afterfocal cerebral ischemia (Botchkina et al., 1997). However, the roleplayed by neuronal TNF-R2 is not clear. In a murine model of retinalischemia, TNF-R2 up-regulation plays a protective role (Fontaine etal., 2002).
The fact that hypoxia, the natural stimulus for peripheral arte-rial chemoreceptors, up-regulates the expression and function ofpro-inflammatory cytokines in the rat CB (Lam et al., 2008), andthat the adaptation to chronic hypoxia involves immune cell inva-sion and increased expression of inflammatory cytokines (Liu et al.,2009), suggests an important cross-talk between oxygen sensingand inflammatory mediators. This interaction could be due, at leastin part, to hypoxia-inducible factor (HIF) activation (Hellwig-Burgelet al., 2005; Frede et al., 2007).
Our novel data, in conjunction with current published evi-dences allow us to propose that the carotid chemoreceptor neuralpathway may also serve as a peripheral sensor for the presenceof immunogenic agents in the blood. Thus, some of the mostprominent manifestations of endotoxemia could be mediated byincoming neural signals from the carotid chemoreceptor pathwayfor immune-to-brain communication.
Acknowledgments
This work was supported by grants DI-02-06/R and DI-39-09/R(to RF), and DI 40-09/R (to FS) from the Division for Research of theUniversidad Andres Bello (UNAB). Thanks are due to Ms. ValentinaSquicciarini and to Mr. Patrick Alvarez, for their assistance duringsome experiments. Special thanks go to Mr. George Montgomeryfor proofreading the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.resp.2010.12.014.
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