LPS Response Research Articles

660 nm Red LED Induces Secretory Leukocyte Protease Inhibitor (SLPI) in Lipopolysaccharide-Stimulated RAW264.7 Cell

SLPI acts as a modulator of the innate immune responses of macrophages, neutrophils and odontoblasts, and LPS-inducible anti-inflammatory cytokine to suppress the production of pro-inflammatory products by macrophages. Many studies have revealed the effects of light emitting diodes (LEDs) on the tissue repair and inflammatory responses. Although the anti-inflammatory mechanisms of irradiation with LEDs in gingival fibroblasts are known, the effects of 660 nm red LEDs on the inflammation remain unclear. Moreover, there is no report regarding the molecular mechanism for the relationship between SLPI and biological effects of LEDs. The effects of 660 nm red LEDs on inflammation with SLPI were investigated by examining the effects of 660 nm LED on the SLPI expression of RAW264.7 cells after LPS stimulation. This paper reports that the 660 nm red LED induced SLPI expression or reduced the LPS response, and inhibited NF-κB activation directly, leading to the suppression of pro-inflammatory cytokines, such as TNF-α and IL-1β, suggesting that it might be a useful wavelength LED for inflammation therapy.

Bovine viral diarrhea virus type 2 in vivo infection modulates TLR4 responsiveness in differentiated myeloid cells which is associated with decreased MyD88 expression

Symptoms of bovine viral diarrhea virus (BVDV) infection range from subclinical to severe, depending on strain virulence. Several in vitro studies showed BVDV infection impaired leukocyte function. Fewer studies have examined the effects of in vivo BVDV infection on monocyte/macrophage function, especially with strains of differing virulence. We characterized cytokine production by bovine myeloid cells isolated early or late in high (HV) or low virulence (LV) BVDV2 infection. Given BVDV infection may enhance susceptibility to secondary bacterial infection, LPS responses were examined as well. Monocytes from HV and LV infected calves produced higher levels of cytokines compared to cells from controls. In contrast, monocyte-derived macrophage cytokine levels were generally reduced. Modulated cytokine expression in HV BVDV2 macrophages was associated with decreased MyD88 expression, likely due to its interaction with viral NS5A. These data and those of others, suggest that certain Flaviviridae may have evolved strategies for subverting receptor signaling pathways involving MyD88.

Investigation of Mf-mediated suppression of B cell activation (TUM10P.1048)

Abstract We have previously shown that C57BL/6J (B/6) peritoneal cavity (PerC) macrophages (Mfs), via IFNg-triggered iNOS expression, reduce T cell activation promoted by mitogens (ConA) or by TCR ligation (anti-CD3e, SEB, etc). We wanted to assess whether PerC B cells were also susceptible to this form of myeloid suppression. B/6 PerC B cell responses triggered by BCR {F(ab’)2 anti-IgM} and TLR4 (LPS/TLR4L) were both suppressed and could be liberated by blocking prostaglandin production with indomethacin; neutralization of IL10 also increased the LPS response. Suppression of the BALB/c (B/c) PerC B cell response to BCR ligation was similar but the TLR4 response was not suppressed. Where the addition of exogenous IL4 or IL13 suppressed the B/6 response to BCR ligation, IL4 increased this response in B/c mice. Most interestingly, the PerC B cell response to CD40 ligation was not suppressed in either strain and addition of IL10 increased this response; addition of IL4 or IL13 suppressed the B/6 response but had no impact upon B/c B cells. Concurrent CD40 and TCR ligation revealed that iNOS-mediated suppression overrides the B cell response. Considering that the high myeloid:lymphoid cell ratio found in PerC cell cultures mimics that seen in tumor microenvironments, these results serve to inform strategies designed to promote anti-cancer immunity.

Central Pathways of MAPKp38 and MAPKJNK Mediate TNFα/iNOS‐Dependent Endotoxic Manifestations of Hypotension and Compromised Heart Rate Variability in Rats

We investigated whether TNFα‐coupled mitogen‐activated protein kinases (MAPKs) in central neurons modulate the early hemodynamic responses evoked by endotoxemia in rats. Changes in blood pressure (BP), heart rate (HR), and HR variability (HRV) caused by i.v. lipopolysaccharide (LPS, 10 mg/kg) were assessed in absence and presence of pharmacologic inhibitors of TNFα, inducible nitric oxide synthase (iNOS), MAPKERK1/2, MAPKp38, or MAPKJNK. Compared with saline, LPS caused significant falls in BP and increases in HR that appeared 30 min post treatment and continued for the following 60 min. The time (standard deviation of beat‐to‐beat intervals, SDNN, and the root mean square of successive beat‐to‐beat differences in R‐R intervals, rMSSD) and frequency domain indices (total power and spectral bands of low and high‐frequency) of HRV were all reduced by LPS. The inhibition of TNFα (pentoxifylline) or iNOS (aminoguanidine) abolished the hemodynamic and HRV effects of LPS. Intracisternal (i.c.) injection of ODQ (guanylate cyclase inhibitor), wortmannin (PI3K inhibitor), SP600125 (MAPKJNK inhibitor) mitigated the LPS‐evoked reductions in BP and HR, but failed to affect the associated decreases in HRV. MAPKp38 inhibition by i.c. SB203580 produced exactly the opposite effects. The reduction in BP or HRV caused by LPS remained unaltered after i.c. PD98059, suggesting no role for MAPKERK1/2 in the LPS responses. Together, the data suggest that central TNFα/iNOS/GC/PI3K pathways linked to MAPKp38 and MAPKJNK are pivotal for provoking the reductions in BP and HRV, respectively, seen during endotoxic shock.

Distinct Mechanisms Regulate IL1B and TNF Gene Expression

We have previously reported that the immediate-early (IE) induction of the genes coding for Interleukin 1β (IL1B) and TNFα (TNF) are regulated by distinct mechanisms. Like many poised IE genes prior to induction, TNF exhibits pre-bound TBP and paused RNA Pol II. In contrast, poised IL1B is devoid of these complexes, revealing only a lineage-specifying factor (Spi-1) and prominent promoter nucleosome depletion. In the case of TNF, LPS treatment releases the paused Pol II for elongation, while for IL1B the activation event results in de novo TBP and Pol II recruitment and elongation. Additionally, once cells are treated with LPS, TNF is rapidly and transiently induced, followed by complete shutdown and is refractory to re-stimulation. For IL1B, a similar rapid-vigorous transient induction is subsequently followed by a lower level of continuous expression, which can be re-induced with LPS. In order to understand the mechanisms involved in these distinct modes of regulation, experiments have been aimed at comparing kinetic RNA expression patterns in the presence of various inducers and inhibitors of transcription. We have observed that in contrast to LPS, phorbol 12-myristate 13-acetate (PMA) generates only the lower level of delayed continuous expression for IL1B, with only a minute effect on TNF. Furthermore, the prolyl hydroxylase activator α-ketoglutarate (α-KG) completely abrogates both the PMA and late/continuous LPS responses, while not affecting TNF. α-KG is a known inhibitor of the HIF-1α transcription factor, which has been previously reported to be important for IL1B gene expression. Our results, suggest that HIF-1α may play a late, rather than immediate-early, role for continuous expression of IL1B.

Gαi1 and Gαi3 regulate macrophage polarization by forming a complex containing CD14 and Gab1

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3 form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3 deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3 knockdown in bone marrow-derived macrophage cells (Gαi1/3 KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3 deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3 were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3 can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.

Expression profiling and functional analysis of Toll-like receptors in primary healthy human nasal epithelial cells shows no correlation and a refractory LPS response.

BackgroundInnate immune recognition via Toll-like receptors (TLRs) on barrier cells like epithelial cells has been shown to influence the regulation of local immune responses. Here we determine expression level variations and functionality of TLRs in nasal epithelial cells from healthy donors.MethodsExpression levels of the different TLRs on primary nasal epithelial cells from healthy donors derived from inferior turbinates was determined by RT-PCR. Functionality of the TLRs was determined by stimulation with the respective ligand and evaluation of released mediators by Luminex ELISA.ResultsPrimary nasal epithelial cells express different levels of TLR1-6 and TLR9. We were unable to detect mRNA of TLR7, TLR8 and TLR10. Stimulation with Poly(I:C) resulted in a significant increased secretion of IL-4, IL-6, RANTES, IP-10, MIP-1β, VEGF, FGF, IL-1RA, IL-2R and G-CSF. Stimulation with PGN only resulted in significant increased production of IL-6, VEGF and IL-1RA. Although the expression of TLR4 and co-stimulatory molecules could be confirmed, primary nasal epithelial cells appeared to be unresponsive to stimulation with LPS. Furthermore, we observed huge individual differences in TLR agonist-induced mediator release, which did not correlate with the respective expression of TLRs.ConclusionOur data suggest that nasal epithelium seems to have developed a delicate system of discrimination and recognition of microbial patterns. Hypo-responsiveness to LPS could provide a mechanism to dampen the inflammatory response in the nasal mucosa in order to avoid a chronic inflammatory response. Individual, differential expression of TLRs on epithelial cells and functionality in terms of released mediators might be a crucial factor in explaining why some people develop allergies to common inhaled antigens, and others do not.

Systemic LPS and inflammatory response during consecutive days of exercise in heat.

This investigation studied circulating LPS activity, potential intestinal damage, and the systemic inflammatory response (SIR) during the exercise heat acclimation process. 8 healthy males (Age=24±3 years) ran in a hot environment on 5 consecutive days until core temperature (Tc) was elevated 2°C above rest. Plasma was obtained pre-, post-, 1 h post-, and 3 h post-exercise on the 1(st), 3(rd), and 5(th) day of exercise and analyzed for TNF-α, IL-6, IL-10, IL-1ra, LPS, and intestinal fatty acid-binding protein (I-FABP). Plasma LPS (1.1 EU·ml(-1)±0.1 vs. 0.7 EU·ml(-1)±0.03; P<0.01) and I-FABP (930.7 pg·ml(-1)±149.0 vs. 640.2 pg·ml(-1)±125.0; P<0.001) were significantly increased post-exercise each. The SIR remained largely unchanged during the study except for TNF-α. Plasma TNF-α was significantly lower on Day 5 at 1 h (3.2 pg·ml(-1)±0.6 vs. 4.5 pg·ml(-1)±0.8; P=0.01) and 3 h (3.6 pg·ml(-1)±0.8 vs. 4.8 pg·ml(-1)±0.9; P=0.05) post-exercise as compared to Day 1. Findings indicate that adaptations to exercise in the heat resulting in reductions of intestinal damage and plasma LPS activity require longer time periods in moderately trained males.

High-Resolution Sequencing and Modeling Identifies Distinct Dynamic RNA Regulatory Strategies

Cells control dynamic transitions in transcript levels by regulating transcription, processing, and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level; editing sites; and transcription, processing, and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Noncanonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one noncanonical strategy. Applying DRiLL to the regulation of noncoding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and posttranscriptional events that control dynamic changes in transcript levels using RNA sequencing data.

Differential requirements of MyD88 and TRIF pathways in TLR4-mediated immune responses in murine B cells

LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety of immune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon-β (TRIF) pathways, but relatively few studies have examined these pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses in B cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88−/− B cells were markedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF−/− B cells were also impaired in these responses compared with WT B cells, but showed better responses than MyD88−/− B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88−/− B cells showed similar patterns of CSR to WT B cells. However, TRIF−/− B cells showed the impaired in the CSR. Compared with WT and MyD88−/− B cells, TRIF−/− B cells exhibited reduced cell division, fewer IgG1+ cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expression in response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization was impaired in TRIF−/− mice, while MyD88−/− mice exhibited increased IgG1 production. Thus, MyD88 and TRIF pathways differently regulate TLR4-induced immune responses in B cells.

Antigen-specific interferon-gamma responses and innate cytokine balance in TB-IRIS.

Background Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB patients receiving antiretroviral therapy (ART). TB-IRIS could be associated with an exaggerated immune response to TB-antigens. We compared the recovery of IFNγ responses to recall and TB-antigens and explored in vitro innate cytokine production in TB-IRIS patients. Methods In a prospective cohort study of HIV-TB co-infected patients treated for TB before ART initiation, we compared 18 patients who developed TB-IRIS with 18 non-IRIS controls matched for age, sex and CD4 count. We analyzed IFNγ ELISpot responses to CMV, influenza, TB and LPS before ART and during TB-IRIS. CMV and LPS stimulated ELISpot supernatants were subsequently evaluated for production of IL-12p70, IL-6, TNFα and IL-10 by Luminex. Results Before ART, all responses were similar between TB-IRIS patients and non-IRIS controls. During TB-IRIS, IFNγ responses to TB and influenza antigens were comparable between TB-IRIS patients and non-IRIS controls, but responses to CMV and LPS remained significantly lower in TB-IRIS patients. Production of innate cytokines was similar between TB-IRIS patients and non-IRIS controls. However, upon LPS stimulation, IL-6/IL-10 and TNFα/IL-10 ratios were increased in TB-IRIS patients compared to non-IRIS controls. Conclusion TB-IRIS patients did not display excessive IFNγ responses to TB-antigens. In contrast, the reconstitution of CMV and LPS responses was delayed in the TB-IRIS group. For LPS, this was linked with a pro-inflammatory shift in the innate cytokine balance. These data are in support of a prominent role of the innate immune system in TB-IRIS.

Elk-3 is a KLF4-regulated gene that modulates the phagocytosis of bacteria by macrophages.

ETS family proteins play a role in immune responses. A unique member of this family, Elk-3, is a transcriptional repressor that regulates the expression of HO-1. Elk-3 is very sensitive to the effects of inflammatory mediators and is down-regulated by bacterial endotoxin (LPS). In the present study, exposure of mouse macrophages to Escherichia coli LPS resulted in decreased, full-length, and splice-variant isoforms of Elk-3. We isolated the Elk-3 promoter and demonstrated that LPS also decreased promoter activity. The Elk-3 promoter contains GC-rich regions that are putative binding sites for zinc-finger transcription factors, such as Sp1 and KLFs. Mutation of the GC-rich region from bp -613 to -603 blunted LPS-induced down-regulation of the Elk-3 promoter. Similar to the LPS response, coexpression of KLF4 led to repression of Elk-3 promoter activity, whereas coexpression of Sp1 increased activity. ChIP assays revealed that KLF4 binding to the Elk-3 promoter was increased by LPS exposure, and Sp1 binding was decreased. Thus, down-regulation of Elk-3 by bacterial LPS is regulated, in part, by the transcriptional repressor KLF4. Overexpression of Elk-3, in the presence of E. coli bacteria, resulted in decreased macrophage phagocytosis. To determine whether limited expression of HO-1 may contribute to this response, we exposed HO-1-deficient bone marrow-derived macrophages to E. coli and found a comparable reduction in bacterial phagocytosis. These data suggest that down-regulation of Elk-3 and the subsequent induction of HO-1 are important for macrophage function during the inflammatory response to infection.

Gut-Homing Conventional Plasmablasts and CD27− Plasmablasts Elicited after a Short Time of Exposure to an Oral Live-Attenuated Shigella Vaccine Candidate in Humans

Currently, there is no licensed Shigella vaccine; however, various promising live-attenuated vaccine candidates have emerged, including CVD1208S (ΔguaBA, Δset, Δsen S. flexneri 2a), which was shown to be safe and immunogenic in Phase 1 clinical trials. Here, we report the immune responses elicited in an outpatient Phase 2 clinical trial in which subjects were vaccinated with CVD 1208S. Oral immunization with CVD 1208S elicited high anti-S. flexneri 2a LPS and IpaB antibody responses as well as an acute plasmablast (PB) infiltration in peripheral blood 7 days after immunization. PB sorted based on their expression of homing molecules confirmed that cells expressing integrin α4β7 alone or in combination with CD62L were responsible for antibody production (as measured by ELISpot). Furthermore, using high-color flow-cytometry, on day 7 after immunization, we observed the appearance of conventional PB (CPB, CD19dim CD20− CD27+high CD38+high CD3−), as well as a PB population that did not express CD27 (CD27− PB; pre-plasmablasts). The pattern of individual or simultaneous expression of homing markers (integrin α4β7, CD62L, CXCR3, and CXCR4) suggested that CPB cells homed preferentially to the inflamed gut mucosa. In contrast, ~50% CD27− PB cells appear to home to yet to be identified peripheral lymphoid organs or were in a transition state preceding integrin α4β7 upregulation. In sum, these observations demonstrate that strong immune responses, including distinct PB subsets with the potential to home to the gut and other secondary lymphoid organs, can be elicited after a short time of exposure to a shigella oral vaccine.

FRI0169 Elevated LBP Levels and in Vivo Activation of Monocytes as Potential Indicators of Bacterial Translocation in Axial Spondyloarthritis

BackgroundSubclinical gut inflammation is often present in axSpA patients [1, 2]. This may result in enhanced translocation of mucosal antigens leading to activation of monocytes which are very sensitive sensors...

Irgm1 regulates the pulmonary innate immune response (MUC5P.864)

Abstract Irgm1 is an IFN-inducible GTPase that mediates host resistance to a variety of intracellular pathogens, and also modulates the LPS response in cultured macrophages. Whether Irgm1 regulates innate immune lung responses and controls host defense against extracellular pathogens is undetermined. We hypothesized that Irgm1 would regulate host defense against extracellular bacteria that cause pneumonia. Compared to wildtype, naïve Irgm1-/- mice had elevated expression of cytokines and chemokines in the lung parenchyma and bronchoalveolar lavage fluid, consistent with steady state inflammation. Moreover, naïve Irgm1-/- lungs displayed abnormal bronchiolocentric lymphocytic infiltration. Compared to wildtype, Irgm1-/- mice had increased influx of PMNs into the airspace after exposure to LPS and S. pneumoniae, but not K. pneumoniae. Bronchoalveolar lavage fluid chemokines, cytokines, and protein concentration (marker of microvascular injury) were all higher in Irgm1-/- mice after LPS and S. pneumonia. Irgm1-/- mice had lower lung CFUs after infection with S. pneumoniae, indicating enhanced pathogen clearance. Irgm1-/- mice also had increased survival after lung infection with S. pneumoniae but not K. pneumonaie. In conclusion, Irgm1-/- mice have an enhanced lung response to LPS and S. pneumoniae, but not K. pneumonaie, implicating a selective role for Irgm1 in host defense. Whether these responses derive from the Irgm1-/- basal lung phenotype is yet to be determined.

Erratum to: LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model

The original publication of this article unfortunately contained an error in Tables 1, 2 and Fig. 3. In both tables published, the last column on the right [LPS doses (ng/ml)] does not include the complete data for the rows. In Fig. 3, parts of the panel a and d [(a) left y-axis is IL-6 (ng/ml); (d) left y-axis is MIP-1beta/CCL4 (pg/ml)] appear hidden. The corrected tables and figure are given below.

LPS and IL‐1 differentially activate mouse and human astrocytes: Role of CD14

Treatment of cultures with toll-like receptor (TLR) ligands or cytokines has become a popular approach to investigate astrocyte neuroinflammatory responses and to simulate the neural environment in various CNS disorders. However, despite much effort, the mechanism of astrocyte activation such as their responses to the TLR ligands and IL-1 remain highly debated. We compared highly pure primary mouse and human astrocyte cultures in their ability to produce proinflammatory mediators (termed "A1") and immunoregulatory mediators (termed "A2") in response to LPS, poly IC, and IL-1 stimulation. In human astrocytes, IL-1 induced both A1 and A2 responses, poly IC induced mostly A2, and LPS induced neither. In mouse astrocytes, LPS induced mostly an A1-predominant response, poly IC induced both A1 and A2, and IL-1 neither. In addition, mouse astrocytes produce abundant IL-1 protein, whereas human astrocytes did not, despite robust IL-1 mRNA expression. Of the TLR4 receptor complex proteins, human astrocytes expressed TLR4 and MD2 but not CD14, whereas mouse astrocytes expressed all three. Mouse astrocyte CD14 (cell-associated and soluble) was potently upregulated by LPS. Silencing TLR4 or CD14 by siRNA suppressed LPS responses in mouse astrocytes. In vivo, astrocytes in LPS-injected mouse brains also expressed CD14. Our results show striking differences between human and mouse astrocytes in the use of TLR/IL-1R and subsequent downstream signaling and immune activation. IL-1 translational block in human astrocytes may be a built-in mechanism to prevent autocrine and paracrine cell activation and neuroinflammation. These results have important implications for translational research of human CNS diseases.

APOε4 is associated with enhanced in vivo innate immune responses in human subjects

The genetic determinants of the human innate immune response are poorly understood. Apolipoprotein (Apo) E, a lipid-trafficking protein that affects inflammation, has well-described wild-type (ε3) and disease-associated (ε2 and ε4) alleles, but its connection to human innate immunity is undefined. We sought to define the relationship of APOε4 to the human innate immune response. We evaluated APOε4 in several functional models of the human innate immune response, including intravenous LPS challenge in human subjects, and assessed APOε4 association to organ injury in patients with severe sepsis, a disease driven by dysregulated innate immunity. Whole blood from healthy APOε3/APOε4 volunteers induced higher cytokine levels on exvivo stimulation with Toll-like receptor (TLR) 2, TLR4, or TLR5 ligands than blood from APOε3/APOε3 patients, whereas TLR7/8 responses were similar. This was associated with increased lipid rafts in APOε3/APOε4 monocytes. By contrast, APOε3/APOε3 and APOε3/APOε4 serum neutralized LPS equivalently and supported similar LPS responses in Apoe-deficient macrophages, arguing against a differential role for secretory APOE4 protein. After intravenous LPS, APOε3/APOε4 patients had higher hyperthermia and plasma TNF-α levels and earlier plasma IL-6 than APOε3/APOε3 patients. APOE4-targeted replacement mice displayed enhanced hypothermia, plasma cytokines, and hepatic injury and altered splenic lymphocyte apoptosis after systemic LPS compared with APOE3 counterparts. In a cohort of 828 patients with severe sepsis, APOε4 was associated with increased coagulation system failure among European American patients. APOε4 is a determinant of the human innate immune response to multiple TLR ligands and associates with altered patterns of organ injury in human sepsis.

Influence of NleH effector expression, host genetics, and inflammation on Citrobacter rodentium colonization of mice

The Escherichia coli NleH1 and NleH2 virulence proteins differentially regulate host transcription of innate immunity genes. The mouse pathogen Citrobacter rodentium encodes one NleH protein, which functions equivalently to E. coli NleH1. We examined the impact of host genetics and intestinal inflammation on the contribution of NleH to C. rodentium colonization of mice differing in LPS responsiveness. NleH expression was detrimental to C. rodentium in C57BL/10ScNJ mice, which do not mount LPS-induced inflammatory responses. This phenotype was reversed if inflammation was induced by chemical means. C. rodentium that expressed both E. coli NleH1 and NleH2 was hypervirulent in C3H/HeJ mice.

The Role of Ets2 Transcription Factor in the Induction of MicroRNA-155 (miR-155) by Lipopolysaccharide and Its Targeting by Interleukin-10

MicroRNA-155 (miR-155) is highly expressed in many cancers such as B cell lymphomas and myeloid leukemia and inflammatory disorders such as rheumatoid arthritis, atopic dermatitis, and multiple sclerosis. The role of miR-155 as both a promoter of inflammation and an oncogenic agent provides a clear need for miR-155 itself to be stringently regulated. We therefore investigated the transcriptional regulation of miR-155 in response to the respective pro- and anti-inflammatory mediators LPS and IL-10. Bioinformatic analysis revealed Ets binding sites on the miR-155 promoter, and we found that Ets2 is critical for miR-155 induction by LPS. Truncation and mutational analysis of the miR-155 promoter confirmed the role of the Ets2 binding site proximal to the transcription start site for LPS responsiveness. We observed increased binding of Ets2 to the miR-155 promoter and Ets2 deficient mice displayed decreased induction of miR-155 in response to LPS. IL-10 inhibited the induction of Ets2 mRNA and protein by LPS, thereby decreasing Ets2 function on the pri-155 promoter. We have thus identified Ets2 as a key novel regulator in both the positive and negative control of miR-155 in the inflammatory response.