TLR4-KO Mice Research Articles

Suppression of Medullar Erythropoiesis in Response to Bacterial Lipopolysaccharides (LPS) Involves Two Distinct TLR4-Dependent Mechanisms with Contrasted Requirements for G-CSF Receptors

Erythropoiesis is a highly controlled process partly regulated in erythroblastic islands by the central erythroblastic island macrophage (EI MΦ) which provides iron, growth factors and mediates enucleation of the maturing erythroblasts. As macrophages are key effectors of inflammation, we investigated the effect of bacterial LPS in vivo on erythropoiesis and EI MΦ defined as CD11b+ F4/80+ VCAM1+ CD169+Ly6G+ in mice. C57BL/6 mice were injected i.p. with 2.5 mg/kg/day LPS from E. coli for 2 days and the effect on medullar erythropoiesis examined at various time-points after this. LPS administration caused a marked whitening of the bone marrow (BM) with decreased numbers of basophilic (9-fold), polychromatic (3.7-fold), orthochromatic erythroblasts (2.2-fold) and reticulocytes (2.5-fold) 48hrs after LPS challenge. Those remained significantly reduced up to 6 days post-LPS. Likewise, EI MΦ were suppressed in the BM 24-48hrs after LPS challenge and remained significantly reduced 6 days post-LPS. This loss of medullar erythropoiesis was compensated by increased number of EI MΦ (13.6-fold), pro-erythroblasts (1.5-fold), polychromatic (1.9-fold), orthochromatic (3.2-fold) and reticulocytes (2.3-fold) in the spleen. As this phenotype resembled suppression of medullar erythropoiesis following G-CSF treatment, we examined whether the mechanism could be indirect via endogenous G-CSF release. LPS induced a transient 80-fold increase in G-CSF concentration in the blood from 123pg/mL (n=6) to 10ng/ml (n=6) 2 days post-LPS. LPS was administered to TLR4 KO and G-CSF receptor (GCSFR) KO mice. These LPS-mediated responses were abrogated in TLR4 KO mice demonstrating that erythropoiesis suppression in response to LPS is fully TLR4-dependant. However responses in GCSFR KO mice were more contrasted. EI MΦ numbers did not change in GCSFR KO mice in response to LPS demonstrating that suppression of EI MΦ in response to LPS is an indirect effect of endogenous G-CSF release. In contrast, medullar erythropoiesis was still suppressed in GCSFR KO mice with significantly reduced numbers of basophilic, polychromatic and orthochromatic erythroblasts demonstrating that medullar erythroblast suppression 1) persists despite the presence of EI MΦ, and 2) is not G-CSF-dependent. Unexpectedly, the BM from GCSFR KO mice treated with LPS was not whitened with high numbers of reticulocytes/erythrocytes. To further understand how BM erythrocytes could be increased whilst erythropoiesis is suppressed in LPS-treated GCSFR KO mice, we measured vascular leakage by injecting Evans Blue i.v. Blood plasma volume in the BM of LPS-treated GCSFR KO mice was 2.9-fold higher compared to LPS-treated wild-type mice and untreated WT and KO mice (6.0±2.0 µL vs 2.1±0.9 µL blood plasma/femur, p=0.005) suggesting that GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS. In conclusion LPS-mediated medullar erythropoiesis suppression involves at least two different TLR4-dependent mechanisms in regards to their requirement for GCSFR: 1) GCSFR-dependent suppression of EI MΦ, 2) GCSFR-independent and EI MΦ-independent suppression of maturing erythroblasts. We are currently investigating whether the latter mechanism involves hepcidin. Finally we also discovered that GCSFR-mediated signaling is necessary to maintain the BM vasculature integrity following LPS challenge. DisclosuresWinkler:GlycoMimetics: Research Funding. Levesque:GlycoMimetics: Equity Ownership.

Lipopolysaccharides-Induced Suppression of Innate-Like B Cell Apoptosis Is Enhanced by CpG Oligodeoxynucleotide and Requires Toll-Like Receptors 2 and 4.

Innate-like B lymphocytes play an important role in innate immunity in periodontal disease through Toll-like receptor (TLR) signaling. However, it is unknown how innate-like B cell apoptosis is affected by the periodontal infection-associated innate signals. This study is to determine the effects of two major TLR ligands, lipopolysaccharide (LPS) and CpG-oligodeoxynucleotides (CpG-ODN), on innate-like B cell apoptosis. Spleen B cells were isolated from wild type (WT), TLR2 knockout (KO) and TLR4 KO mice and cultured with E. coli LPS alone, P. gingivalis LPS alone, or combined with CpG-ODN for 2 days. B cell apoptosis and expressions of specific apoptosis-related genes were analyzed by flow cytometry and real-time PCR respectively. P. gingivalis LPS, but not E. coli LPS, reduced the percentage of AnnexinV+/7-AAD- cells within IgMhighCD23lowCD43-CD93- marginal zone (MZ) B cell sub-population and IgMhighCD23lowCD43+CD93+ innate response activator (IRA) B cell sub-population in WT but not TLR2KO or TLR4KO mice. CpG-ODN combined with P. gingivalis LPS further reduced the percentage of AnnexinV+/7-AAD- cells within MZ B cells and IRA B cells in WT but not TLR2 KO or TLR4 KO mice. Pro-apoptotic CASP4, CASP9 and Dapk1 were significantly down-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT but not TLR2 KO or TLR4 KO mice. Anti-apoptotic IL-10 was significantly up-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT and TLR2 KO but not TLR4 KO mice. These results suggested that both TLR2 and TLR4 signaling are required for P. gingivalis LPS-induced, CpG-ODN-enhanced suppression of innate-like B cell apoptosis.

LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development.

Immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota activate retinal microglia in mouse models.

Although its actual role in the progression of degenerative processes is not fully known, the persistent activated state of retinal microglia and the concurrent secretion of inflammatory mediators may contribute to neuronal death and permanent vision loss. Our objective was to determine whether non-ocular conditions (immunosuppression and peripheral inflammation) could lead to activation of retinal microglia. Mouse models of immunosuppression induced by cyclophosphamide and/or peripheral inflammation by chemically induced sublethal colitis in C57BL/6J mice were used. Retinal microglia morphology, spatial distribution and complexity, as well as MHCII and CD11b expression levels were determined by flow cytometry and confocal immunofluorescence analysis with anti-CD11b, anti-IBA1 and anti-MHCIIRT1B antibodies. Retinas of mice with double treatment showed changes in microglial morphology, spatial distribution and expression levels of CD11b and MHCII. These effects were higher than those observed with any treatment separately. In addition, we also observed in these mice: (i) translocation of endogenous bacteria from gut to liver, and (ii) upregulation of TLR2 expression in retinal microglia. Using a mouse model of immunosuppression and gut colonization by Candida albicans, translocation of fungal cells was confirmed to occur in wild type and, to a higher extent, in TLR2 KO mice, which are more susceptible to fungal invasion; interestingly microglial changes were also higher in TLR2 KO mice. Hence, non-ocular injuries (immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota) can activate retinal microglia and therefore could affect the progression of neurodegenerative disorders and should be taken into account to improve therapeutic options.

Elevated CD14 (Cluster of Differentiation 14) and Toll-Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice.

Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc.

TRAP-positive osteoclast precursors mediate ROS/NO-dependent bactericidal activity via TLR4

Osteoclastogenesis was induced by RANKL stimulation in mouse monocytes to examine the possible bactericidal function of osteoclast precursors (OCp) and mature osteoclasts (OCm) relative to their production of NO and ROS. Tartrate-resistant acid phosphatase (TRAP)-positive OCp, but few or no OCm, phagocytized and killed Escherichia coli in association with the production of reactive oxygen species (ROS) and nitric oxide (NO). Phagocytosis of E. coli and production of ROS and NO were significantly lower in TRAP+ OCp derived from Toll-like receptor (TLR)-4 KO mice than that derived from wild-type (WT) or TLR2-KO mice. Interestingly, after phagocytosis, TRAP+ OCp derived from wild-type and TLR2-KO mice did not differentiate into OCm, even with continuous exposure to RANKL. In contrast, E. coli-phagocytized TRAP+ OCp from TLR4-KO mice could differentiate into OCm. Importantly, neither NO nor ROS produced by TRAP+ OCp appeared to be engaged in phagocytosis-induced suppression of osteoclastogenesis. These results suggested that TLR4 signaling not only induces ROS and NO production to kill phagocytized bacteria, but also interrupts OCm differentiation. Thus, it can be concluded that TRAP+ OCp, but not OCm, can mediate bactericidal activity via phagocytosis accompanied by the production of ROS and NO via TLR4-associated reprograming toward phagocytic cell type.

Abstract 417: Control of Adipose Tissue Macrophage Metabolism by Oxidized Phospholipids

Our group has previously shown that oxidized phospholipids (OxPL) induce a unique macrophage (MΦ) phenotype known as “Mox,” distinct from pro- and anti-inflammatory M1 and M2 phenotypes. We have also shown that Mox make up 30% of the MΦs found in atherosclerotic lesions. OxPL induce TLR2-dependent inflammatory gene expression in MΦs. TLR2 activation by peptidoglycan was shown to induce accumulation of ceramides, which can alter bioenergetics by inhibiting the mitochondria. Recently, it has been shown that spleen tyrosine kinase (Syk) is phosphorylated upon TLR2 agonist Pam3CSK stimulation in MΦs. The effect of OxPL on bioenergetics has never before been studied. Here we test the hypothesis that OxPL change MΦ bioenergetics and inflammatory capacity via a TLR2-Syk-Ceramide pathway. Using flow cytometry, we found that in mice fed a high-fat diet, more than 20% of all adipose tissue MΦs can be described as Mox. Concomitantly, using immunohistochemistry and liquid-chromatography mass spectrometry, we measured increased levels of OxPL in the stromal vascular fraction of obese murine adipose tissue, as compared to lean controls. We treated bone marrow-derived MΦs (BMDMs) from WT, TLR2-KO, and Syk-KO mice with OxPL and measured bioenergetics using a Seahorse Flux Analyzer. Our results show that OxPL decrease oxygen consumption rate (OCR), a measure of oxidative phosphorylation, and decrease extracellular acidification rate (ECAR), a measure of glycolytic capacity. These MΦs can be described as quiescent, but they still engage in low-level cytokine production. Moreover, OxPL result in the accumulation of ceramides, as quantified by mass spectrometry. Finally, using Syk-KO MΦs and Syk inhibitors, we show that OxPL-induced inflammatory gene expression and ceramide accumulation are dependent on Syk. In summary, our results demonstrate that OxPL accumulate in obese adipose tissue and induce a change in the inflammatory and metabolic profiles of MΦs, involving a TLR2-ceramide-Syk dependent pathway. These results suggest that OxPL are triggers of adipose tissue inflammation and subsequent development of insulin resistance. Furthermore, we identify Syk as a therapeutic target for inhibiting diet-induced adipose tissue inflammation and insulin resistance.

TLR4 signaling mediates AP-1 activation in an MPTP-induced mouse model of Parkinson's disease

ObjectiveTo evaluate the effects of Toll-like receptor 4 (TLR4) signaling on the activation of the transcription factor activator protein-1 (AP-1) in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease (PD). MethodsThe following groups were evaluated: normal saline (NS)-treated WT mice, NS-treated TLR4-knockout (KO) mice, MPTP-treated WT mice, and MPTP-treated TLR4-KO mice. After establishing the mouse model, behavioral changes were evaluated. AP-1 expression was detected by RT-PCR, Western blotting, immunohistochemistry and immunofluorescence staining. ResultsCompared to MPTP-treated WT mice, significantly reduced dyskinesia was observed in MPTP-treated TLR4-KO mice. AP-1 mRNA and protein levels were significantly up-regulated in the substantia nigras (SNs) of MPTP-treated WT mice relative to NS-treated mice (P<0.01); these levels were significantly reduced in MPTP-treated TLR4-KO mice relative to MPTP-treated WT mice (P<0.01). Immunohistochemical staining demonstrated that AP-1 was distributed throughout the SN in MPTP-treated mice, and immunofluorescence further showed that AP-1 was expressed in TH-positive neuronal cells and GFAP-positive astrocytes. In addition, immunofluorescence revealed that AP-1 expression was lower in TH-positive neurons and GFAP-positive astrocytes in the SNs of MPTP-treated TLR4-KO mice relative to MPTP-treated WT mice. ConclusionsThe TLR4 pathway may play an important role in regulating AP-1 activation.

Abstract 12161: Toll-like Receptor 9 Plays a Pivotal Role in Angiotensin II-induced Atherosclerosis

Background: Chronic vascular inflammation causes atherosclerosis. Toll-like receptor (TLR) 9 recognizes bacterial unmethylated DNA and plays a role in innate defense, whereas it can also provoke inflammation in response to fragmented DNA released from damaged mammalian cells. However, the role of TLR9 in the development of atherosclerosis remains unknown. Here, we investigated whether genetic deletion of TLR9 attenuates atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. Methods and Results: We generated TLR9/ApoE double knockout (dKO) mice by crossing ApoE KO mice and TLR9 KO mice, and infused angiotensin II (Ang II) (1000 ng/kg/min) subcutaneously for 28 days using osmotic pump. There were no differences in blood pressure and plasma lipid levels between ApoE KO mice and TLR9/ApoE dKO mice. Ang II infusion significantly increased plasma levels of single-stranded DNA and double-stranded DNA, endogenous ligands of TLR9, in both strains of mice (P&lt;0.001, respectively). TLR9/ApoE dKO mice showed less atherosclerotic lesion progression in the aortic arch and less lipid accumulation in atherosclerotic plaques in the aortic root compared with ApoE KO mice (P&lt;0.05, respectively). TLR9/ApoE dKO mice also showed less gene expression of inflammatory molecules (e.g., ICAM-1, MCP-1) in the abdominal aorta. The result of cell sorting experiment using atherosclerotic aorta obtained from Ang II-infused ApoE KO mice demonstrated that macrophage population mainly expressed TLR9 in this tissue. We, therefore, performed in vitro experiments using thioglycolate-stimulated peritoneal macrophages. CpG ODN, agonistic oligonucleotide for TLR9, markedly promoted the expression of inflammatory molecules (e.g., ICAM-1 and MCP-1) in wild-type macrophages but not in TLR9 KO macrophages. Furthermore, conditioned medium (CM) of human umbilical vein endothelial cells (HUVECs) treated with Ang II promoted gene expression of ICAM-1 and TNF-α in THP-1 macrophages compared with CM of non-treated HUVECs. Conclusion: Our results suggested that Ang II infusion activates the TLR9 signaling in macrophages, leading to the promotion of vascular inflammation and atherosclerosis. TLR9 may serve as a potential therapeutic target for atherosclerotic diseases.

Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation

Necrotic cells passively release HMGB1, which can stimulate TLR4 in an autocrine fashion to potentially initiate “sterile” inflammation that maintains different disease states. We have shown that prooxidants can induce NF-κB activation through TLR4 stimulation. We examined whether prooxidants enhance HMGB1-induced TLR4 signaling through NF-κB activation. We used LPS-EK as a specific agonist for TLR4, and PPC and SIN-1 as in situ sources for ROS. As model systems, we used HEK-Blue cells (stably transfected with mouse TLR4), RAW-Blue™ cells (derived from murine RAW 264.7 macrophages) and primary murine macrophages from TLR4-KO mice. Both HEK-Blue and RAW-Blue 264.7 cells express optimized secreted embryonic alkaline phosphatase (SEAP) reporter under the control of a promoter inducible by NF-κB. We treated cells with HMGB1 alone and/or in conjunction with prooxidants and/or inhibitors using SEAP release as a measure of TLR4 stimulation. HMGB1 alone and/or in conjunction with prooxidants increased TNFα and IL-6 released from TLR4-WT, but not from TLR4-KO macrophages. Pro-oxidants increased HMGB1 release, which we quantified by ELISA. We used both fluorescence microscopy imaging and flow cytometry to quantify the expression of intracellular ROS. TLR4-neutralizing antibody decreased prooxidant-induced HMGB1 release. Prooxidants promoted HMGB1-induced NF-κB activation as determined by increased release of SEAP and TNF-α, and accumulation of iROS. HMGB1 (Box A), anti-HMGB1 and anti-TLR4-neutralizing pAbs inhibited HMGB1-induced NF-κB activation, but HMGB1 (Box A) and anti-HMGB1 pAb had no effect on prooxidant-induced SEAP release. The present results confirm that prooxidants enhance proinflammatory effects of HMGB1 by activating NF-κB through TLR4 signaling.

TLR9 Mediates Remote Liver Injury following Severe Renal Ischemia Reperfusion.

Ischemia reperfusion injury is a common cause of acute kidney injury and is characterized by tubular damage. Mitochondrial DNA is released upon severe tissue injury and can act as a damage-associated molecular pattern via the innate immune receptor TLR9. Here, we investigated the role of TLR9 in the context of moderate or severe renal ischemia reperfusion injury using wild-type C57BL/6 mice or TLR9KO mice. Moderate renal ischemia induced renal dysfunction but did not decrease animal well-being and was not regulated by TLR9. In contrast, severe renal ischemia decreased animal well-being and survival in wild-type mice after respectively one or five days of reperfusion. TLR9 deficiency improved animal well-being and survival. TLR9 deficiency did not reduce renal inflammation or tubular necrosis. Rather, severe renal ischemia induced hepatic injury as seen by increased plasma ALAT and ASAT levels and focal hepatic necrosis which was prevented by TLR9 deficiency and correlated with reduced circulating mitochondrial DNA levels and plasma LDH. We conclude that TLR9 does not mediate renal dysfunction following either moderate or severe renal ischemia. In contrast, our data indicates that TLR9 is an important mediator of hepatic injury secondary to ischemic acute kidney injury.

Toll-like receptor 4-induced endoplasmic reticulum stress contributes to impairment of vasodilator action of insulin.

Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Activation of Toll-like receptor 4 (TLR4) induces proinflammatory response and endoplasmic reticulum (ER) stress. Saturated fatty acids (SFA) activate TLR4, which induces ER stress and endothelial dysfunction. Therefore, we determined whether TLR4-mediated ER stress is an obligatory step mediating SFA-induced endothelial dysfunction. Palmitate stimulated proinflammatory responses and ER stress, and this was suppressed by knockdown of TLR4 in primary human aortic endothelial cells (HAEC). Next, we examined the role of TLR4 in vasodilatory responses in intact vessels isolated from wild-type (WT, C57BL/6) and TLR4-KO mice after feeding high-fat (HFD) or normal chow diet (NCD) for 12 wk. Arterioles isolated from HFD WT mice exhibited impaired insulin-stimulated vasodilation compared with arterioles isolated from NCD WT mice. Deficiency of TLR4 was protective from HFD-induced impairment of insulin-stimulated vasodilation. There were no differences in acetylcholine (Ach)- or sodium nitroprusside (SNP)-stimulated vasodilation between the two groups. Furthermore, we examined whether ER stress is involved in SFA-induced impairment of vasodilator actions of insulin. Infusion of palmitate showed the impairment of vasodilatory response to insulin, which was ameliorated by coinfusion with tauroursodeoxycholic acid (TUDCA), an ER stress suppressor. Taken together, the results suggest that TLR4-induced ER stress may be an obligatory step mediating the SFA-mediated endothelial dysfunction.

Abstract P030: Early Cognitive Deficits Induced by Diabetes Are Masked by an Increase in Exploratory Behavior: Evidence From TLR2 Knock-out Mice

Toll-like receptor 2 (TLR2) has been shown to contribute to cardiovascular complications of diabetes such as nephropathy. However, the role of TLR2 in cerebrovascular inflammation and dysfunction leading to cognitive impairment, an emerging cerebrovascular complication of diabetes and hypertension, remains unknown. We reported that streptozotocin (STZ)-induced type-1 diabetic TLR2 knockout mice were protected from decreased cerebral blood flow (CBF) at 4 and 6 weeks post induction. Given that decreased CBF precedes cognitive impairment, we hypothesized that these TLR2-KO STZ mice would subsequently be protected from cognitive impairment. 10 week old male C57Bl:6 and TLR2-KO mice were injected with 50mg STZ/kg body weight daily for 5 days and observed alongside control mice. Fasting blood glucose was measured, with levels &gt; 240 mg/dL considered to confirm diabetes. Cognitive function was assessed via Y-maze testing. There was no difference in total arm entries between WT and WT-STZ mice or between WT and TLR2-KO. There was a significant decrease in total entries between TLR2-KO and TLR2-KO STZ (40.33± 1.88 vs.29.67± 2.09, p≥0.05, n=12/group). However, percent novel arm entries was statistically non-significant amongst all groups. Paradoxically, both diabetic groups had a non-significant increase in percent of time spent in the novel arm vs. their respective controls (WT-STZ: 35.7±5.3 seconds vs. WT: 33.2± 4.4 seconds; TLR2-KO STZ: 41.6±5.8 seconds vs. TLR2-KO: 37.9±5.5 seconds). The lack of difference in percent novel arm entries is likely a result of the relative increase in exploratory behavior in the STZ groups. Additional experiments with open-field testing and novel object recognition test are being performed to determine if the apparent lack of change in short term hippocampal memory is due to a confounding artifact of the exploration time and whether these cognitive tests are accompanied by changes in CBF in this cohort. If indeed TLR2 KO mice are protected from early mild cognitive deficits, TLR2 antagonism may be a novel target in the prevention/treatment of cerebrovascular complications of diabetes.

Proneness of TLR5 deficient mice to develop colitis is microbiota dependent

Alterations in the gut microbiota have been implicated to play a role in potentiating inflammatory bowel diseases in both humans and mice. Mice lacking the flagellin receptor, toll-like receptor 5 (TLR5), are prone to develop spontaneous gut inflammation, but are significantly protected when treated with antibiotics or maintained in germ-free conditions. However, given that the incidence of spontaneous inflammation in TLR5KO mice is quite variable in conventional conditions (typically ∼10% show clear colitis), this result is far from definitive and does not rule out that TLR5KO mice might be prone to develop inflammation even in the absence of a microbiota. Herein, we demonstrate that neutralization of IL10 signaling induces colitis in 100% of TLR5KO mice which provide a more rigorous approach to evaluate the role of microbiota in gut inflammation. Mice treated with antibiotics or maintained in germ-free condition are substantially protected against IL-10R neutralization-induced colitis, underscoring that gut inflammation in TLR5KO mice is dependent upon the presence of a gut microbiota.

High-fat diet induces toll-like receptor 4-dependent macrophage/microglial cell activation and retinal impairment.

The toll-like receptor 4 (TLR4) signaling pathway is involved in chronic inflammation and insulin resistance, which are associated with obesity and diabetes mellitus. In the present study, a model of high-fat diet (HFD) feeding of mice was used to investigate the role of TLR4 in overnutrition- and obesity-associated inflammation and infiltration of macrophages and microglia in the retina. Wild-type C57BL/6 and TLR4 knockout (TLR4KO; B6.B10ScN-Tlr4(lps-del)/JthJ) mice were fed a HFD or control chow diet (CD) for 6 months. The TLR4 expression, the relative increase in macrophages/microglia (CD11b(+) and CD45(+) cells), the presence of markers of oxidative stress (gp91phox and malondialdehyde; MDA), and DNA damage (phosphorylated histone H2AX; γH2AX) were assessed by real-time PCR and immunofluorescence studies. The HFD for 6 months showed increased obesity, glucose intolerance, and insulin resistance in mice. Toll-like receptor 4 expression was found in vascular pericytes at the inner retina. Increased CD11b(+) and CD45(+) cells, phosphorylated NF-κB, interleukin-6, gp91phox, MDA, and γH2AX were observed in the retina of mice fed a HFD compared to CD counterparts. TLR4KO mice did not show the adverse effects of HFD. Our results indicate that HFD-induced macrophage/microglial cell activation and retinal impairment were reduced in the absence of TLR4. The findings suggest that TLR4 is implicated in the pathogenesis of retinal diseases caused by metabolic disorders.

Role of Toll like receptor 4 signaling pathway in the secondary damage induced by experimental spinal cord injury

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is specific immunologic response to systemic bacterial infection and injury. TLRs contribute to the initial induction of neuroinflammation in the CNS. In spinal cord injury (SCI) intricate immune cell interactions are triggered, typically consisting of a staggered multiphasic immune cell response, which can become deregulated. The present study aims to evaluate the role of TLR4 signaling pathway in the development of secondary damage in a mouse model of SCI using TLR4-deficient (TLR4-KO) mice such as C57BL/10ScNJ and C3H/HeJ mice. We evaluated behavioral changes, histological, immunohistochemistry and molecular assessment in TLR4-KO after SCI. SCI was performed on TLR4-KO and wild-type (WT) mice by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. Mice were sacrificed at 24h after SCI to evaluate the various parameters. SCI TLR4 KO mice developed severer hind limb motor dysfunction and neuronal death by histological evaluation, myeloid differentiation primary response 88 (Myd88) expression as well as an increase in nuclear factor NF-κB activity, tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels, glial fibrillary acidic protein (GFAP), microglia marker (CD11β), inducible nitric oxide synthases (iNOS), poly-ADP-ribose polymerase (PARP) and nitrotyrosine expression compared to WT mice. Moreover, the absence of TLR4 also caused a decrease in phosphorylated interferon regulatory transcription factor (p-IRF3) and interferon (IFN-β) release. In addition, SCI TLR4 KO mice showed in spinal cord tissues a more pronounced up-regulation of Bax and a down-regulation of Bcl-2 compared to SCI WT mice. Finally, we clearly demonstrated that TLR4 is important for coordinating post-injury sequel and in regulating inflammation after SCI.

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin

RationaleRespiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly.ObjectivesTo assess the effect of Gram-positive bacterial infection on fibrosis...

A novel recombinant adjuvant replacing the CD4+ T helper cell functions in inducing IgG isotype antibodies and protection by T cell dependent influenza split vaccines (VAC8P.1052)

Abstract The action mechanisms of adjuvants have not been well understood yet. We hypothesized that an effective adjuvant would overcome a defect in CD4+ T cell help for the induction of immune responses and protection by T cell dependent vaccines. We found that current influenza split vaccine was not able to induce antibodies and protection in a CD4+ T cell deficient condition. In an attempt to develop an effective adjuvant, we developed a recombinant virus-like particle expressing flagellin (FliC-VLP) and investigated its adjuvant effects and mechanisms by comparing alum adjuvant. CD4 knockout (CD4KO) mice that were immunized with influenza split vaccine plus FliC-VLP (or MF59, AS04 human adjuvant) induced IgG isotype-switched antibodies, germinal center formation, and virus-specific memory cells, all of which were not induced in CD4KO mice with split vaccine alone. Levels of antibodies and protective efficacy in CD4KO mice with split vaccine plus FliC-VLP were significantly higher than those in C57BL/6 wild type mice without adjuvant and CD4KO mice adjuvanted by alum. The adjuvanticity of FliC-VLP was partially dependent on TLR5 when tested in TLR5KO mice. Taken together, the results provide evidence that FliC-VLP adjuvant can stimulate the induction of antigen-specific immune responses and improve protection against lethal infection in CD4KO mice immunized with T cell dependent split vaccines, indicating CD4+ T cell independent action mechanisms of adjuvant.

In Vivo Role of TLR2 and MyD88 Signaling in Eliciting Innate Immune Responses in Staphylococcal Endophthalmitis.

The purpose of this study was to investigate the protective mechanisms evoked by TLR2 and MyD88 signaling in bacterial endophthalmitis in vivo. Endophthalmitis was induced in wild-type (WT), TLR2(-/-), MyD88(-/-), and Cnlp(-/-) mice by intravitreal injections of a laboratory strain (RN6390) and two endophthalmitis isolates of Staphylococcus aureus. Disease progression was monitored by assessing corneal and vitreous haze, bacterial burden, and retinal tissue damage. Levels of inflammatory cytokines/chemokines were determined using quantitative RT-PCR (qRT-PCR) and ELISA. Flow cytometry was used to assess neutrophil infiltration. Cathelicidin-related antimicrobial peptide (CRAMP) expression was determined by immunostaining and dot blot. Eyes infected with either laboratory or clinical isolates exhibited higher levels of inflammatory mediators at the early stages of infection (≤24 hours) in WT mice than in TLR2(-/-) or MyD88(-/-) mice. However, their levels surpassed that of WT mice at the later stages of infection (>48 hours), coinciding with increased bacterial burden and retinal damage. Both TLR2(-/-) and MyD88(-/-) retinas produced reduced levels of CRAMP, and its deficiency (Cnlp(-/-)) rendered the mice susceptible to increased bacterial burden and retinal tissue damage as early as 1 day post infection. Analyses of inflammatory mediators and neutrophil levels in WT versus Cnlp(-/-) mice showed a trend similar to that observed in TLR2 and MyD88 KO mice. Furthermore, we observed that even a 10-fold lower infective dose of S. aureus was sufficient to cause endophthalmitis in TLR2(-/-) and MyD88(-/-) mice. TLR2 and MyD88 signaling plays an important role in protecting the retina from staphylococcal endophthalmitis by production of the antimicrobial peptide CRAMP.

Toll-like Receptor 2 is Dispensable for an Immediate-early Microglial Reaction to Two-photon Laser-induced Cortical Injury In vivo.

Microglia, the resident macrophages in the central nervous system, can rapidly respond to pathological insults. Toll-like receptor 2 (TLR2) is a pattern recognition receptor that plays a fundamental role in pathogen recognition and activation of innate immunity. Although many previous studies have suggested that TLR2 contributes to microglial activation and subsequent pathogenesis following brain tissue injury, it is still unclear whether TLR2 has a role in microglia dynamics in the resting state or in immediate-early reaction to the injury in vivo. By using in vivo two-photon microscopy imaging and Cx3cr1GFP/+ mouse line, we first monitored the motility of microglial processes (i.e. the rate of extension and retraction) in the somatosensory cortex of living TLR2-KO and WT mice; Microglial processes in TLR2-KO mice show the similar motility to that of WT mice. We further found that microglia rapidly extend their processes to the site of local tissue injury induced by a two-photon laser ablation and that such microglial response to the brain injury was similar between WT and TLR2-KO mice. These results indicate that there are no differences in the behavior of microglial processes between TLR2-KO mice and WT mice when microglia is in the resting state or encounters local injury. Thus, TLR2 might not be essential for immediate-early microglial response to brain tissue injury in vivo.