Recombinant TLR5 Agonist CBLB502 Promotes NK Cell-Mediated Anti-CMV Immunity in Mice

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.     

High Fat Diet-Induced Gut Microbiota Exacerbates Inflammation and Obesity in Mice via the TLR4 Signaling Pathway

Background & Aims

While it is widely accepted that obesity is associated with low-grade systemic inflammation, the molecular origin of the inflammation remains unknown. Here, we investigated the effect of endotoxin-induced inflammation via TLR4 signaling pathway at both systemic and intestinal levels in response to a high-fat diet.

Methods

C57BL/6J and TLR4-deficient C57BL/10ScNJ mice were maintained on a low-fat (10 kcal % fat) diet (LFD) or a high–fat (60 kcal % fat) diet (HFD) for 8 weeks.

Results

HFD induced macrophage infiltration and inflammation in the adipose tissue, as well as an increase in the circulating proinflammatory cytokines. HFD increased both plasma and fecal endotoxin levels and resulted in dysregulation of the gut microbiota by increasing the Firmicutes to Bacteriodetes ratio. HFD induced the growth of Enterobecteriaceae and the production of endotoxin in vitro. Furthermore, HFD induced colonic inflammation, including the increased expression of proinflammatory cytokines, the induction of Toll-like receptor 4 (TLR4), iNOS, COX-2, and the activation of NF-κB in the colon. HFD reduced the expression of tight junction-associated proteins claudin-1 and occludin in the colon. HFD mice demonstrated higher levels of Akt and FOXO3 phosphorylation in the colon compared to the LFD mice. While the body weight of HFD-fed mice was significantly increased in both TLR4-deficient and wild type mice, the epididymal fat weight and plasma endotoxin level of HFD-fed TLR4-deficient mice were 69% and 18% of HFD-fed wild type mice, respectively. Furthermore, HFD did not increase the proinflammatory cytokine levels in TLR4-deficient mice.

Conclusions

HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the gut microbiota composition and increasing its intestinal permeability through the induction of TLR4, thereby accelerating obesity.     

IL-23 Induces Atopic Dermatitis-Like Inflammation Instead of Psoriasis-Like Inflammation in CCR2-Deficient Mice

Psoriasis is an immune-mediated chronic inflammatory skin disease, characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. IL-23 is expressed in psoriatic skin, and IL-23 injected into the skin of mice produces IL-22-dependent dermal inflammation and acanthosis. The chemokine receptor CCR2 has been implicated in the pathogenesis of several inflammatory diseases, including psoriasis. CCR2-positive cells and the CCR2 ligand, CCL2 are abundant in psoriatic lesions. To examine the requirement of CCR2 in the development of IL-23-induced cutaneous inflammation, we injected the ears of wild-type (WT) and CCR2-deficient (CCR2^−/−) mice with IL-23. CCR2^−/− mice had increased ear swelling and epidermal thickening, which was correlated with increased cutaneous IL-4 levels and increased numbers of eosinophils within the skin. In addition, TSLP, a cytokine known to promote and amplify T helper cell type 2 (Th2) immune responses, was also increased within the inflamed skin of CCR2^−/− mice. Our data suggest that increased levels of TSLP in CCR2^−/− mice may contribute to the propensity of these mice to develop increased Th2-type immune responses.     

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation

1. Download high-res image (219KB)
2. Download full-size image

     

Telomere-Mediated Chromosomal Instability Triggers TLR4 Induced Inflammation and Death in Mice

Background

Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC^−/− mice) display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR) are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation.

Methodology/Principal Findings

We examined the function of TLR4 in telomerase deficient mTERC^−/− mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC^+/+), mTERC^+/− and mTERC^−/− mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFα and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC^−/−) mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-κB binding to its promoter by down-regulating ATF-3 in mTERC^−/− macrophages.

Conclusions/Significance

Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-κB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups.     

The p53 Tumor Suppressor Causes Congenital Malformations in Rpl24-Deficient Mice and Promotes Their Survival

Hypomorphic mutation in one allele of ribosomal protein l24 gene (Rpl24) is responsible for the Belly Spot and Tail (Bst) mouse, which suffers from defects of the eye, skeleton, and coat pigmentation. It has been hypothesized that these pathological manifestations result exclusively from faulty protein synthesis. We demonstrate here that upregulation of the p53 tumor suppressor during the restricted period of embryonic development significantly contributes to the Bst phenotype. However, in the absence of p53 a large majority of Rpl24^Bst/+ embryos die. We showed that p53 promotes survival of these mice via p21-dependent mechanism. Our results imply that activation of a p53-dependent checkpoint mechanism in response to various ribosomal protein deficiencies might also play a role in the pathogenesis of congenital malformations in humans.Nascent ribosome biogenesis is required during cell growth, proliferation and differentiation (42, 47). It is temporally and spatially organized within the nucleolus, where rRNAs are transcribed, processed, modified, and assembled with ribosomal proteins (RPS) to generate the mature 40S and 60S ribosomal subunits (13). RPS participate in additional steps in ribosome biogenesis in the nucleoplasm and the cytoplasm, such as the transport of ribosomal precursors, stabilization of ribosome structure, and regulation of different steps in protein synthesis (15).The critical role of at least some RPS in mammals is underscored by the pathological or lethal consequences of the deficiency of just one allele. Only a few heterozygous mutations of RP genes have been shown to be viable in mammals, and each of them has shown a relatively specific phenotype. Germ line heterozygous mutation for RPS19, RPS24, RPS17, RPL35a, RPL5, and RPL11 genes have been found in patients with Diamond-Blackfan anemia, which is characterized by absent or decreased erythropoiesis, and less frequently by small stature and various somatic malformations that mostly occur in the cephalic region, as well as an increased incidence of leukemia, osteogenic sarcoma, and myelodisplastic syndrome (7, 9, 12, 17, 18). Recently, a link between heterozygous mutations in Rps19 and Rps20 and dark skin phenotype in mice has been demonstrated (29).Another heterozygous RP mutant is the Belly Spot and Tail (Bst) mouse (34). This is a semidominant, hypomorphic mutation caused by an intronic deletion in the Rpl24 gene, affecting Rpl24 mRNA splicing. Rpl24^Bst/+ mice are characterized by reduced body size, a white ventral middle spot, white hind feet, retinal abnormalities, a kinked tail, and other skeletal abnormalities. Since Rpl24^Bst/+ mouse embryonic fibroblasts from these mice showed a significant reduction in the rate of overall protein synthesis, it has been suggested that their phenotype result exclusively from faulty translation of mRNAs in tissues that depend on rapid and flawless protein synthesis (34).It has been argued that the differential phenotypes of heterozygous mutants of RP genes in mammals might be attributable to the expression levels of the respective RP and the consequent decrease in the amount of ribosomes, impairment of specific steps in protein synthesis, and potential extraribosomal function. However, it should be pointed out that the relative contribution of the impaired protein synthesis or extraribosomal function to these phenotypes remains to be determined (9, 17, 34, 36, 37, 43, 57).Recent evidence indicates that deficiencies in individual RPS could lead to pathological consequences via activation of a p53-dependent checkpoint regulatory mechanism. We demonstrated that inducible deletion of the Rps6 gene in the liver of adult mice inhibits the synthesis of the 40S ribosomal subunit, as well as proliferation of liver cells, after partial hepatectomy, despite seemingly unaffected protein synthesis (54). These observations suggested the existence of a novel checkpoint, downstream of the deficiency in ribosome biogenesis. Likewise, the perigastrulation lethality of Rps6 heterozygote embryos appears to reflect the triggering of a p53-dependent checkpoint response rather than a deficit in protein synthesis (37). We have assumed that activation of a p53-dependent checkpoint is triggered by impaired rRNA processing in the nucleolus in Rps6-deficient cells, since the nucleolar structure and function are compromised by almost all known p53-inducing stresses (37, 41, 50). Based on all of these observations, it could be speculated that the rare occurrence of RP heterozygosity in mammals reflects the fatal consequences of p53-dependent checkpoint activation (36, 37). In addition to function in development, this checkpoint may also play a role in other processes. Since various RP deficiencies in Drosophila melanogaster, zebrafish, and humans pose a great risk for development of malignant tumors, it is possible that induction of a p53-dependent checkpoint response prevents expansion of such potentially hazardous cells (1, 9, 11, 56).Recently, we initiated an RNA interference screen for RP deficiencies that upregulate the p53 tumor suppressor in A549 cells. It has been previously suggested that a defect in ribosome biogenesis in the nucleolus caused by a RP deficiency triggers the p53 response (36, 37, 50). A number of studies in yeast showed that Rpl24 does not participate in ribosome biogenesis in the nucleolus, but it assembles late with the nascent 60S ribosomes in the cytoplasm and regulates the 60S subunit joining step during translation initiation and other steps in protein synthesis (10, 25, 45). Thus, it was surprising to observe that RPL24 deficiency triggered the p53 response in our screen. This observation led us to consider the possibility that p53 is upregulated in Rpl24^Bst/+ mice. In contrast to previous opinion that the phenotype of these mice results exclusively from impaired protein synthesis (34), we demonstrate here that it is largely caused by the aberrant upregulation of p53 protein expression during embryonic development.     

Association of TLR4 and Treg in Helicobacter pylori Colonization and Inflammation in Mice

The host immune response plays an important role in the pathogenesis of Helicobacter pylori infection. The aim of this study was to clarify the immune pathogenic mechanism of Helicobacter pylori infection via TLR signaling and gastric mucosal Treg cells in mice. To discover the underlying mechanism, we selectively blocked the TLR signaling pathway and subpopulations of regulatory T cells in the gastric mucosa of mice, and examined the consequences on H. pylori infection and inflammatory response as measured by MyD88, NF-κB p65, and Foxp3 protein expression levels and the levels of Th1, Th17 and Th2 cytokines in the gastric mucosa. We determined that blocking TLR4 signaling in H. pylori infected mice decreased the numbers of Th1 and Th17 Treg cells compared to controls (P < 0.001–0.05), depressed the immune response as measured by inflammatory grade (P < 0.05), and enhanced H. pylori colonization (P < 0.05). In contrast, blocking CD25 had the opposite effects, wherein the Th1 and Th17 cell numbers were increased (P < 0.001–0.05), immune response was enhanced (P < 0.05), and H. pylori colonization was inhibited (P < 0.05) compared to the non-blocked group. In both blocked groups, the Th2 cytokine IL-4 remained unchanged, although IL-10 in the CD25 blocked group was significantly decreased (P < 0.05). Furthermore, MyD88, NF-κB p65, and Foxp3 in the non-blocked group were significantly lower than those in the TLR4 blocked group (P < 0.05), but significantly higher than those of the CD25 blocked group (P < 0.05). Together, these results suggest that there might be an interaction between TLR signaling and Treg cells that is important for limiting H. pylori colonization and suppressing the inflammatory response of infected mice.     

Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.     

An Early Reduction in Treg Cells Correlates with Enhanced Local Inflammation in Cutaneous Leishmaniasis in CCR6-Deficient Mice

Resistance to Leishmania major infection is dependent on the development of a cell-mediated Th1 immune response in resistant C57BL/6 mice whereas Th2-prone BALB/c mice develop non-healing lesions after infection. The chemokine receptor CCR6 is shared by anti-inflammatory regulatory T cells and pro-inflammatory Th17 cells. In a recent study we showed that C57BL/6 mice deficient in CCR6 exhibited enhanced footpad swelling and impaired T helper cell migration indicated by reduced recruitment of total T helper cells into the skin after infection and a reduced delayed type hypersensitivity reaction. Based on these findings we tested whether the lack of CCR6 alters Treg or Th17 cell responses during the course of Leishmania major infection. When we analyzed T cell subsets in the lymph nodes of CCR6-deficient mice, Th17 cell numbers were not different. However, reduced numbers of Treg cells paralleled with a stronger IFNγ response. Furthermore, the early increase in IFNγ-producing cells correlated with increased local tissue inflammation at later time points. Our data indicate an important role of CCR6 for Treg cells and a redundant role for Th17 cells in a Th1 cell-driven anti-parasitic immune response against Leishmania major parasites in resistant C57BL/6 mice.     

Helicobacter typhlonius and Helicobacter rodentium Differentially Affect the Severity of Colon Inflammation and Inflammation-Associated Neoplasia in IL10-Deficient Mice

Infection with Helicobacter species is endemic in many animal facilities and may alter the penetrance of inflammatory bowel disease (IBD) phenotypes. However, little is known about the relative pathogenicity of H. typhlonius, H. rodentium, and combined infection in IBD models. We infected adult and neonatal IL10^−/− mice with H. typhlonius, H. rodentium, or both bacteria. The severity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelicobacter therapy. Infected IL10^−/− mice developed IBD with severity of noninfected (minimal to no inflammation) < H. rodentium < H. typhlonius < mixed H. rodentium + H. typhlonius (severe inflammation). Inflammation-associated colonic neoplasia was common in infected mice and its incidence correlated with IBD severity. Combined treatment with amoxicillin, clarithromycin, metronidazole, and omeprazole eradicated Helicobacter in infected mice and ameliorated established IBD in both infected and noninfected mice. Infection of IL10^−/− mice with H. rodentium, H. typhlonius, or both organisms can trigger development of severe IBD that eventually leads to colonic neoplasia. The high incidence and multiplicity of neoplastic lesions in infected mice make this model well-suited for future research related to the development and chemoprevention of inflammation-associated colon cancer. The similar antiinflammatory effect of antibiotic therapy in Helicobacter-infected and -noninfected IL10^−/− mice with colitis indicates that unidentified microbiota in addition to Helicobacter drive the inflammatory process in this model. This finding suggests a complex role for both Helicobacter and other intestinal microbiota in the onset and perpetuation of IBD in these susceptible hosts.Abbreviations: IBD, Inflammatory bowel diseaseInflammatory bowel disease (IBD) is hypothesized to develop due to aberrant immune responses induced by gut microbes.⁵ IBD does not occur in germ-free IL10^−/− mice,^2,15 indicating the importance of microorganisms as environmental triggers of intestinal inflammation. However, conventionally colonized or specific pathogen-free IL10^−/− mice may develop colitis spontaneously^2,32 or in response to specific triggers such as nonsteroidal antiinflammatory drugs^3,14 or infections with certain bacteria.^6,16,18 The normal lack of ongoing immune responses against bacteria in subjects without IBD has been attributed to the immunologic tolerance that specifically downregulates immune responses against antigens derived from these bacteria. Nevertheless, despite a large number of studies, no single bacterial type has fulfilled Koch postulates and been confirmed as a cause of IBD in animals or humans.Previous studies used fluorescence in situ hybridization with probes specific for bacterial 16S rRNA combined with conventional histologic techniques to study the relationships between various species of intestinal bacteria and the mucosa in mice and humans with IBD.^33,34 Those studies showed that in normal mice, most bacterial groups are separated from the mucosal surface by either a mucus layer that excludes bacteria or, in the cecum and proximal colon, by an ‘interlaced’ layer that is composed of tightly packed bacteria. The interlaced or mucus layer thus limits the contact of the bulk of the enteric bacteria with the mucosal epithelium. In contrast, complex biofilms composed of multiple species of bacteria that were firmly adherent to the mucosal surface were identified in the majority of colon tissue samples collected from humans and mice with IBD.^33,34 The presence of a biofilm abrogates the protective effects of the normal layer of mucus and can allow luminal bacterial antigens and toxins to reach the unshielded epithelial surface, where they can trigger cascades of host inflammatory responses. Situations that cause defects in the epithelial surface or degrade the protective qualities of mucus or the interlaced layer (or both) may allow contact of bacterial antigens and adjuvants with immune cells located in the lamina propria and lead to the generation of immune responses that result in IBD.³⁴Helicobacter species are used frequently to model microbial triggers of colon inflammation, because they have previously been linked to the development of both IBD- and inflammation-associated neoplasia.^11,21,29 Most studies have been performed by using Helicobacter hepaticus or H. bilis.²⁰ However, H. typhlonius, H. rodentium, H. muridarum, H. ganmani, H. trogontum and other species^{8,12,17,29,35} can also be endemic in research animal facilities. The pathophysiologic effects of these less-common Helicobacter species are, for the most part, poorly investigated.Most rodent Helicobacter species are urease-negative and therefore preferentially colonize the intestine, but some species produce urease enzyme and can translocate to the liver or colonize the biliary system.¹³ H. typhlonius was shown to cause an enteric disease characterized by mucosal hyperplasia and associated inflammation in the cecum and colon in immunodeficient mice^11,23 and IL10^−/− mice.¹⁸ H. typhlonius is genetically related most closely to H. hepaticus, having only 2.36% difference in the 16S rRNA gene sequence, but H. typhlonius has a unique intervening sequence in this gene that makes it easily recognizable by PCR.^9,12 Molecular detection of this pathogen with PCR is rapid, sensitive and allows the detection of the early phases of infection; further enhanced sensitivity is achieved with nested primers.²² One of the most important features of PCR is that it can be performed noninvasively on fecal pellets. Data regarding the pathogenetic mechanisms of H. rodentium are scarce.^35,36 H. rodentium alone apparently does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr mice; however, coinfection with H. hepaticus and H. rodentium was associated with augmented cecal gene expression and clinical diarrheal disease in immunodeficient mice compared with mice infected with H. hepaticus alone.²³Previous reports demonstrated that H. typhlonius was capable of initiating colitis in adult IL10^−/− mice.^10,11 In those studies, colitis was relatively mild, with no development of inflammation-associated neoplasia. H. rodentium has been described to be nonpathogenic in adult wild-type mice but did enhance cytokine production in the cecum of mice also infected with H. hepaticus.²³ We recently observed rapid onset of severe IBD and a high incidence of inflammation-associated neoplasia in IL10^−/− mice that were coinfected with both H. typhlonius and H. rodentium as pups.¹⁶ The current study was undertaken to determine the relative roles of H. rodentium and H. typhlonius, individually and in combination, and age at infection in the development of colon inflammation and inflammation-associated neoplasia in IL10^−/− mice. Novel features of our model include controlled infection of the combination of H. typhlonius and H. rodentium⁹ and infection of IL10^−/− mice during the neonatal period.     

Protoporphyrin Treatment Modulates Susceptibility to Experimental Autoimmune Encephalomyelitis in miR-155-Deficient Mice

We previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155^-/- CD4⁺ T cells to promote antigen-driven inflammation after adoptive transfer in antigen-expressing recipients. Protoporphyrins are molecules recognized for their modulatory effect on HO-1 expression and function. In the present study, we investigated the effect of protoporphyrin treatment on the development of autoimmunity in miR-155-deficient mice. MiR-155-mediated control of HO-1 expression in promoting T cell-driven chronic autoimmunity was confirmed since HO-1 inhibition restored susceptibility to experimental autoimmune encephalomyelitis (EAE) in miR-155-deficient mice. The increased severity of the disease was accompanied by an enhanced T cell infiltration into the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of chronically-stimulated T cells in EAE.     

Role of the Toll Like Receptor (TLR) Radical Cycle in Chronic Inflammation: Possible Treatments Targeting the TLR4 Pathway

Activation of the Toll-like receptor 4 (TLR4) complex, a receptor of the innate immune system, may underpin the pathophysiology of many human diseases, including asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation. TLRs are pattern recognition receptors that recognize damage-associated molecular patterns and pathogen-associated molecular patterns, including lipopolysaccharide (LPS) from gram-negative bacteria. Here we focus on the environmental factors, which are known to trigger TLR4, e.g., ozone, atmosphere particulate matter, long-lived reactive oxygen intermediate, pentachlorophenol, ionizing radiation, and toluene. Activation of the TLR4 pathways may cause chronic inflammation and increased production of reactive oxygen and nitrogen species (ROS/RNS) and oxidative and nitrosative stress and therefore TLR-related diseases. This implies that drugs or substances that modify these pathways may prevent or improve the abovementioned diseases. Here we review some of the most promising drugs and agents that have the potential to attenuate TLR-mediated inflammation, e.g., anti-LPS strategies that aim to neutralize LPS (synthetic anti-LPS peptides and recombinant factor C) and TLR4/MyD88 antagonists, including eritoran, CyP, EM-163, epigallocatechin-3-gallate, 6-shogaol, cinnamon extract, N-acetylcysteine, melatonin, and molecular hydrogen. The authors posit that activation of the TLR radical (ROS/RNS) cycle is a common pathway underpinning many “civilization” disorders and that targeting the TLR radical cycle may be an effective method to treat many inflammatory disorders.     

Developmental Exposure of Mice to Dioxin Promotes Transgenerational Testicular Inflammation and an Increased Risk of Preterm Birth in Unexposed Mating Partners

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, commonly known as dioxin) is a ubiquitous environmental contaminant and known endocrine disruptor. Using a mouse model, we previously found that adult female mice exposed in utero to TCDD (F1 generation) as well as multiple subsequent generations (F2-F4) exhibited reduced fertility and an increased incidence of spontaneous preterm birth. Additional studies revealed that male F1 mice with a similar in utero/developmental TCDD exposure also exhibited diminished fertility and conferred an increased risk of preterm birth to their unexposed mating partners. Herein, we extend these previous observations, reporting that reduced fertility in male F1 mice is linked to testicular inflammation which coincides with apoptosis of developing spermatocytes, sub-fertility and an increased risk of preterm birth in their unexposed mating partners. Significantly, in the absence of additional toxicant exposure, testicular inflammation and reduced fertility persisted in F2 and F3 males and their control mating partners also frequently exhibited spontaneous preterm birth. Although a steady, global decline in male fertility has been noted over the last few decades, the reasons for these changes have not been firmly established. Likewise, the PTB rate in the U.S. and other countries has paralleled industrial development, suggesting a possible relationship between environmental toxicant exposure and adverse pregnancy outcomes. Most current clinical strategies to prevent preterm birth are focused solely on the mother and have yielded limited benefits. In contrast, our studies strongly suggest that the preconception testicular health of the father is a critical determinant of pregnancy outcomes in mice. Future clinical studies should examine the potential contribution of the male to gestation length in women and whether efforts to reduce the incidence of preterm birth should be initiated in both parents prior to pregnancy.     

Effects of Sleep Fragmentation and Chronic Latent Viral Infection on Behavior and Inflammation in Mice

Many chronic diseases are associated with both fatigue and disrupted or nonrestorative sleep. In addition, so-called ‘sickness behaviors’ (for example, anorexia, anhedonia, reduced social interaction, fatigue) are common during infectious and inflammatory disease and have been linked to facets of the immune response. To study these relationships, we used murine gammaherpesvirus (MuGHV), a natural pathogen of wild rodents that provides an experimental model for studying the pathophysiology of an Epstein-Barr (EBV)-like γ-herpesvirus infection in mice. We exposed male and female C57BL/6J mice that were either uninfected or latently infected with MuGHV to either sleep fragmentation (SF) or control conditions and measured the effects on behavior and markers of inflammation. Exposure of infected male mice to SF during the normal somnolent (light) phase significantly reduced locomotor activity during the subsequent active phase, despite an intervening 6-h rest period. Infection was associated with significant increases in lung IFNγ and CXC motif ligand (CXCL) 10 in both male and female mice. In both infected and uninfected male mice, exposure to SF was associated with lower levels of IL1β and C-C motif ligand (CCL) 3 in lung. Exposure of infected female mice to SF led to reductions in lung IL2, CXCL1, and CCL 3. Thus, compared with control conditions, SF was generally associated with lower concentrations of various cytokines in lung. These findings, together with our previous work, indicate that complex interactions among several host factors likely contribute to the behavioral and inflammatory changes associated with viral infection and sleep disruption even in a well-controlled mouse model.Abbreviations: CCL, CC motif ligand; CXCL, CXC motif ligand; CW, consolidated walking; DE, disk environment; EBV, Epstein–Barr virus; HC, home cage; MuGHV, murine gammaherpesvius; SF, sleep fragmentationSo-called ‘sickness behaviors’ (for example, fatigue, anorexia, anhedonia, reduced social interaction) have often been linked to facets of the host response to immune or inflammatory challenge. During chronic infections or other inflammatory conditions, the continually primed host immune response is likely to create a powerful and unrelenting stimulus for these debilitating symptoms in some patients. Epstein–Barr virus (EBV) is a ubiquitous human gammaherpesvirus that causes acute disease, establishes life-long latency, and is associated with the common syndrome of infectious mononucleosis and with other conditions. Infection with or reactivation of EBV in humans is often associated with fatigue and excessive sleepiness.^{1,20,24,46,53} These and related symptoms could be mediated in part by immune activation or dysfunction, neural–endocrine homeostatic imbalance, or both, as produced secondary to the acute and chronic viral infection.^7,36 Studies demonstrating that antibodies to latent EBV proteins are elevated in some patients with chronic fatigue have led to speculation that 1) the synthesis of these viral proteins reflects stress-related reactivation of latent virus,^17,23,31 and 2) although host responses to reactivation may abort complete viral replication, the viral proteins themselves or the resultant host immune response may trigger or exacerbate fatigue.¹⁸Because gammaherpesviruses coevolved with their host species, they are highly species-specific, and infections of animals with EBV have rarely been reported. However, murine gammaherpesvirus (MuGHV) is a natural pathogen of wild rodents that provides an experimental model for studying the pathophysiology of an EBV-like gammaherpesvirus in laboratory mice, which are a permissive host.^15,16,32 After intranasal infection of immunocompetent mice with MuGHV, pulmonary viral titers peak between days 6 to 9, and lytic virus is cleared from the lung in about 10 to 14 d.¹⁴ Like other herpesviruses, MuGHV can maintain lifelong latency in its host. Latent virus is established in lung within days after infection.¹⁴ Spleen, lymph nodes, and bone marrow also harbor latent virus, primarily in B lymphocytes.^13-15,44 The number of latently infected cells peaks at approximately day 14 after infection and then declines rapidly, such that viral load at 4 wk after infection is barely above the limits of detection.⁶ Immunologic features of MuGHV infection resemble the infectious mononucleosis stage of EBV infection.^5,9,15,51 Key common features of EBV and MuGHV infections include splenomegaly and lymphocytosis.Our past work has shown that mice develop significant clinical illness during the acute stage of MuGHV infection and show elevated behavioral sensitivity to challenge with LPS during the chronic–latent stage of infection.³³ In our more recent work,⁴⁹ male C57BL/6J and BALB/cByJ mice that were placed in a novel cage containing its normal inhabitant during the initial 6 h of the light (somnolent) phase were less active during the subsequent dark (active) phase, independent of infection status and despite a 6-h light-phase interval that provided time for recovery sleep after the period of socially induced arousal. In these mice, changes in inflammatory mediators were complex, with both independent and interactive effects of infection status, mouse strain, and exposure to the perturbation.⁴⁹ However, the chemokine CXC motif ligand (CXCL) 10 was consistently elevated in the lung of latently infected mice. Exposure to an unfamiliar mouse for 6 h also was associated with higher pulmonary concentrations of CXCL10, IL1β, granulocyte colony-stimulating factor, and monocyte chemoattractant protein 1 at 12 h after exposure, independent of infection status. These data indicate that exposure of mice to a social challenge is associated with 1) a pulmonary inflammatory response and 2) reduced activity that could represent fatigue, depression, or other facets of sickness behavior; both of these effects are at least in part independent of mouse strain and latent MuGHV infection.To further investigate the effect of common mild stressors on behavior and inflammation in association with latent viral infection, we exposed mice to sleep fragmentation (SF). We selected SF as a perturbation in light of the common incidence of disturbed or shortened sleep in the human population. In our laboratory, we induce SF by placing mice on a rotating disk that requires them to engage in frequent but brief epochs of walking. Therefore, we also evaluated 2 control conditions: consolidated walking (CW) and exposure to the disk environment with minimal rotation (DE).     

Monocyte Chemoattractant Protein-1 Deficiency Attenuates Oxidative Stress and Protects against Ovariectomy-Induced Chronic Inflammation in Mice

Background

Loss of ovarian function is highly associated with an elevated risk of metabolic disease. Monocyte chemoattractant protein-1 (MCP-1, C-C chemokine ligand 2) plays critical roles in the development of inflammation, but its role in ovariectomy (OVX)-induced metabolic disturbance has not been known.

Methodology and Principal Findings

We investigated the role of MCP-1 in OVX-induced metabolic perturbation using MCP-1-knockout mice. OVX increased fat mass, serum levels of MCP-1, macrophage-colony stimulating factor (M-CSF), and reactive oxygen species (ROS), whereas MCP-1 deficiency attenuated these. OVX-induced increases of visceral fat resulted in elevated levels of highly inflammatory CD11c-expressing cells as well as other immune cells in adipose tissue, whereas a lack of MCP-1 significantly reduced all of these levels. MCP-1 deficiency attenuated activation of phospholipase Cγ2, transforming oncogene from Ak strain, and extracellular signal-regulated kinase as well as generation of ROS, which is required for up-regulating CD11c expression upon M-CSF stimulation in bone marrow-derived macrophages.

Conclusions/Significance

Our data suggested that MCP-1 plays a key role in developing metabolic perturbation caused by a loss of ovarian functions through elevating CD11c expression via ROS generation.     

Lacticaseibacillus rhamnosus LS8 Ameliorates Azoxymethane/Dextran Sulfate Sodium-Induced Colitis-Associated Tumorigenesis in Mice via Regulating Gut Microbiota and Inhibiting Inflammation

Probiotics and Antimicrobial Proteins - Gut microbiota dysbiosis may promote the process of colorectal cancer (CRC). Lacticaseibacillus rhamnosus LS8 (LRL) is a potential gut microbiota regulating...     

Early Transplantation of Bone Marrow Mononuclear Cells Promotes Neuroprotection and Modulation of Inflammation After Status Epilepticus in Mice by Paracrine Mechanisms

Status epilepticus (SE) is a severe clinical manifestation of epilepsy associated with intense neuronal loss and inflammation, two key factors involved in the pathophysiology of temporal lobe epilepsy. Bone marrow mononuclear cells (BMMC) attenuated the consequences of pilocarpine-induced SE, including neuronal loss, in addition to frequency and duration of seizures. Here we investigated the effects of BMMC transplanted early after the onset of SE in mice, as well as the involvement of soluble factors produced by BMMC in the effects of the cell therapy. Mice were injected with pilocarpine for SE induction and randomized into three groups: transplanted intravenously with 1 × 10⁷ BMMC isolated from GFP transgenic mice, injected with BMMC lysate, and saline-treated controls. Cell tracking, neuronal counting in hippocampal subfields and cytokine analysis in the serum and brain were performed. BMMC were found in the brain 4 h following transplantation and their numbers progressively decreased until 24 h following transplantation. A reduction in hippocampal neuronal loss after SE was found in mice treated with live BMMC and BMMC lysate when compared to saline-treated, SE-induced mice. Moreover, the expression of inflammatory cytokines IL-1β, TNF-α, IL-6 was decreased after injection of live BMMC and to a lesser extent, of BMMC lysate, when compared to SE-induced controls. In contrast, IL-10 expression was increased. Analysis of markers for microglia activation demonstrated a reduction of the expression of genes related to type 1-activation. BMMC transplantation promotes neuroprotection and mediates anti-inflammatory effects following SE in mice, possibly through the secretion of soluble factors.