Homotrimeric Macrophage Migration Inhibitory Factor (MIF) Drives Inflammatory Responses in the Corneal Epithelium by Promoting Caveolin-rich Platform Assembly in Response to Infection

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa–induced IL-8 responses via the formation of caveolin-1-rich “signaling hubs” in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.     

Surfactant Protein D Contributes to Ocular Defense against Pseudomonas aeruginosa in a Murine Model of Dry Eye Disease

Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10⁹ cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10–14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.     

Microbiota-Independent Ameliorative Effects of Antibiotics on Spontaneous Th2-Associated Pathology of the Small Intestine

We have previously generated a mouse model of spontaneous Th2-associated disease of the small intestine called TRAF6ΔDC, in which dendritic cell (DC)-intrinsic expression of the signaling mediator TRAF6 is ablated. Interestingly, broad-spectrum antibiotic treatment ameliorates TRAF6ΔDC disease, implying a role for commensal microbiota in disease development. However, the relationship between the drug effects and commensal microbiota status remains to be formally demonstrated. To directly assess this relationship, we have now generated TRAF6ΔDC bone marrow chimera mice under germ-free (GF) conditions lacking commensal microbiota, and found, unexpectedly, that Th2-associated disease is actually exacerbated in GF TRAF6ΔDC mice compared to specific pathogen-free (SPF) TRAF6ΔDC mice. At the same time, broad-spectrum antibiotic treatment of GF TRAF6ΔDC mice has an ameliorative effect similar to that observed in antibiotics-treated SPF TRAF6ΔDC mice, implying a commensal microbiota-independent effect of broad-spectrum antibiotic treatment. We further found that treatment of GF TRAF6ΔDC mice with broad-spectrum antibiotics increases Foxp3⁺ Treg populations in lymphoid organs and the small intestine, pointing to a possible mechanism by which treatment may directly exert an immunomodulatory effect. To investigate links between the exacerbated phenotype of the small intestines of GF TRAF6ΔDC mice and local microbiota, we performed microbiotic profiling of the luminal contents specifically within the small intestines of diseased TRAF6ΔDC mice, and, when compared to co-housed control mice, found significantly increased total bacterial content characterized by specific increases in Firmicutes Lactobacillus species. These data suggest a protective effect of Firmicutes Lactobacillus against the spontaneous Th2-related inflammation of the small intestine of the TRAF6ΔDC model, and may represent a potential mechanism for related disease phenotypes.     

Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection

Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host’s defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.     

Patterns of early gut colonization shape future immune responses of the host

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system.     

Tacrolimus (FK506) Suppresses TREM-1 Expression at an Early but Not at a Late Stage in a Murine Model of Fungal Keratitis

Purpose

To investigate the efficacy and mechanism of tacrolimus(FK506), which is a novel macrolide immunosuppressant, in inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) expression in a murine keratitis model induced by Aspergillus fumigatus.

Method

TREM-1 was detected in 11 fungus-infected human corneas by quantitative real-time PCR (qRT-PCR). RAW264.7 macrophages were divided into four groups, which received treatment with zymosan (100 µg/ml), zymosan (100 µg/ml) + mTREM-1/Fc protein (1 µg/ml), or zymosan (100 µg/ml) + FK506 (20 µM) or negative-control treatment. After this treatment, the expression of TREM-1, interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) was assayed using qRT-PCR and ELISA. The mouse model of fungal keratitis was created by intrastromal injection with Aspergillus fumigatus, and the mice were divided into 2 groups: group A received vehicle eye drops 4 times each day, and group B received 4 doses of FK506 eye drops each day. Corneal damage was evaluated by clinical scoring and histologic examination,and myeloperoxidase (MPO) protein levels were also detected by ELISA. The expression of TREM-1, IL-1β and TNFα was then determined at different time points using qRT-PCR and ELISA.

Results

TREM-1 expression dramatically increased in the human corneas with fungal keratitis. In contrast, FK506 reduced the expression of TREM-1, IL-1β and TNFα in RAW264.7 macrophages stimulated with zymosan. In the mouse model, at day 1 post-infection, the corneal score of the FK506-treated group was lower than that of the control, and polymorphonuclear neutrophil (PMN) infiltration was diminished. TREM-1, IL-1β and TNFα expression was significantly reduced at the same time point. However, the statistically significant differences in cytokine expression, clinical scores and infiltration disappeared at 5 days post-infection.

Conclusions

FK506 may inhibit the inflammation induced by fungi and alleviate the severity of corneal damage at an early stage of fungal keratitis by downregulating TREM-1 expression.     

Ascorbic acid deficiency induces hepatic and intestinal expression of inflammation-related genes irrespective of the presence or absence of gut microbiota in ODS rats

We have previously demonstrated that ascorbic acid (AsA) deficiency causes inflammatory changes in the liver and intestine in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. We have suggested that AsA deficiency increased intestinal interleukine (IL)-6 production, stimulating hepatic acute phase proteins (APPs) expression via the portal vein. In this study, we determined whether these hepatic and intestinal inflammatory changes by AsA deficiency are induced in germ-free (GF) ODS rats. For 18 days, male specific pathogen-free (SPF) ODS rats were fed the basal diet containing 600 mg AsA/kg (control group) or the AsA-free diet (AsA-deficient group) in SPF conditions, while male GF ODS rats were fed the basal diet (control group) or the AsA-free diet (AsA-deficient group) in GF conditions. Firstly, AsA deficiency significantly elevated the hepatic expression of APPs in both SPF and GF rats. In hepatic mRNA levels of some APPs, significant interaction between GF and AsA-deficiency effects was observed. Secondly, AsA deficiency elevated intestinal IL-6 and IL-1β mRNA levels in both SPF and GF rats, and significant interaction between GF and AsA-deficiency effects was observed in these mRNA levels of jejunum and cecum. In SPF and GF rats, AsA deficiency elevated portal IL-6 concentration. These results show that AsA deficiency caused hepatic and intestinal inflammatory changes in both the GF and SPF ODS rats and indicate that AsA deficiency could directly induce intestinal inflammatory changes without the involvement of gut microbiota.     

The Gut Microbiota and Developmental Programming of the Testis in Mice

Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.     

Intestinal Microbiota Regulate Xenobiotic Metabolism in the Liver

Background

The liver is the central organ for xenobiotic metabolism (XM) and is regulated by nuclear receptors such as CAR and PXR, which control the metabolism of drugs. Here we report that gut microbiota influences liver gene expression and alters xenobiotic metabolism in animals exposed to barbiturates.

Principal findings

By comparing hepatic gene expression on microarrays from germfree (GF) and conventionally-raised mice (SPF), we identified a cluster of 112 differentially expressed target genes predominantly connected to xenobiotic metabolism and pathways inhibiting RXR function. These findings were functionally validated by exposing GF and SPF mice to pentobarbital which confirmed that xenobiotic metabolism in GF mice is significantly more efficient (shorter time of anesthesia) when compared to the SPF group.

Conclusion

Our data demonstrate that gut microbiota modulates hepatic gene expression and function by altering its xenobiotic response to drugs without direct contact with the liver.     

Role of the indigenous microbiota in maintaining the virus-specific CD8 memory T cells in the lung of mice infected with murine cytomegalovirus

The potent role of indigenous microbiota in maintaining murine CMV (MCMV)-specific memory T cells, which were measured by multimer staining, was investigated using germfree (GF) mice. When the BALB/c mice bred under specific pathogen-free (SPF) conditions were i.p. infected with 0.2 LD(50) of MCMV, high frequencies of CD69(+)/CD44(+) MCMV-specific CD8 T cells were noted in the lungs even at 6-12 mo after infection (11.1 +/- 3.2 and 9.8 +/- 0.9%, respectively). In contrast, even though the viral load and expression levels of mRNA of such cytokines as IL-2, IL-7, IL-15, and IFN-gamma in the lungs of MCMV-infected GF mice were comparable to those of infected SPF mice, the frequencies of MCMV-specific CD8 T cells in the lungs of infected GF mice were kept lower than 1% at 6-12 mo after infection. In addition, the reconstitution of microbiota of MCMV-infected GF mice by orally administering a fecal suspension prepared from SPF mice restored the frequencies of both CD8(+)/multimer(+) and CD8(+)/multimer(-) T cells to levels similar to those found in SPF mice. These results suggested the indigenous microbiota to play a crucial role in the expansion and maintenance of viral-specific CD8 memory T cells, probably by cross-reactivity between the antigenic epitope of the MCMV-specific memory T cells and the variety of peptides derived from the members of the microbiota. Such cross-reactivity may thus be a major feature of those cells.     

CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14^−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.     

A Multi-Center,Cross-Sectional Study on the Burden of Infectious Keratitis in China

Objective

To understand the prevalence and demographic characteristics of infectious keratitis and infectious corneal blindness.

Methods

A multi-center, population-based cross-sectional study was conducted from January 1 to August 31, 2010. A total of 191,242 individuals of all age groups from 10 geographically representative provinces were sampled using stratified, multi-stage, random and systematic sampling procedures. A majority, 168,673 (88.2%), of those sampled participated in the study. The examination protocol included a structured interview, visual acuity testing, an external eye examination, and an anterior segment examination using a slit lamp. The causes and sequelae of corneal disease were identified using uniform customized protocols. Blindness in one eye caused by infectious keratitis was defined as infectious corneal blindness.

Results

The prevalence of past and active infectious keratitis was 0.192% (95% confidence interval [CI], 0.171–0.213%), and the prevalence of viral, bacterial, and fungal keratitis was 0.11%, 0.075%, and 0.007%, respectively. There were 138 cases of infectious corneal blindness in at least one eye in the study population (prevalence of 0.082% [95%CI, 0.068%–0.095%]). Statistical analysis suggested that ocular trauma, alcoholic consumption, low socioeconomic levels, advanced age, and poor education were risk factors for infectious corneal blindness.

Conclusions

Infectious keratitis is the leading cause of corneal blindness in China. Eye care strategies should focus on the prevention and rehabilitation of infectious corneal blindness.     

Interactions between multiple helminths and the gut microbiota in wild rodents

The gut microbiota is vital to host health and, as such, it is important to elucidate the mechanisms altering its composition and diversity. Intestinal helminths are host immunomodulators and have evolved both temporally and spatially in close association with the gut microbiota, resulting in potential mechanistic interplay. Host–helminth and host–microbiota interactions are comparatively well-examined, unlike microbiota–helminth relationships, which typically focus on experimental infection with a single helminth species in laboratory animals. Here, in addition to a review of the literature on helminth–microbiota interactions, we examined empirically the association between microbiota diversity and composition and natural infection of multiple helminth species in wild mice (Apodemus flavicollis), using 16S rRNA gene catalogues (metataxonomics). In general, helminth presence is linked with high microbiota diversity, which may confer health benefits to the host. Within our wild rodent system variation in the composition and abundance of gut microbial taxa associated with helminths was specific to each helminth species and occurred both up- and downstream of a given helminth''s niche (gut position). The most pronounced helminth–microbiota association was between the presence of tapeworms in the small intestine and increased S24–7 (Bacteroidetes) family in the stomach. Helminths clearly have the potential to alter gut homeostasis. Free-living rodents with a diverse helminth community offer a useful model system that enables both correlative (this study) and manipulative inference to elucidate helminth–microbiota interactions.     

Identification of causative pathogens in mouse eyes with bacterial keratitis by sequence analysis of 16S rDNA libraries

The clone library method using PCR amplification of the 16S ribosomal RNA (rRNA) gene was used to identify pathogens from corneal scrapings of C57BL/6-corneal opacity (B6-Co) mice with bacterial keratitis. All 10 samples from the eyes with bacterial keratitis showed positive PCR results. All 10 samples from the normal cornea showed negative PCR results. In all 10 PCR-positive samples, the predominant and second most predominant species accounted for 20.9 to 40.6% and 14.7 to 26.1%, respectively, of each clone library. The predominant species were Staphylococcus lentus, Pseudomonas aeruginosa, and Staphylococcus epidermidis. The microbiota analysis detected a diverse group of microbiota in the eyes of B6-Co mice with bacterial keratitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The bacterial species detected in this study were mostly in accordance with results of studies on clinical bacterial keratitis in human eyes. Based on the results of our previous studies and this study, the B6-Co mouse should be considered a favorable model for studying bacterial keratitis.     

CD36/SR-B2-TLR2 Dependent Pathways Enhance Porphyromonas gingivalis Mediated Atherosclerosis in the Ldlr KO Mouse Model

There is strong epidemiological association between periodontal disease and cardiovascular disease but underlying mechanisms remain ill-defined. Because the human periodontal disease pathogen, Porphyromonas gingivalis (Pg), interacts with innate immune receptors Toll-like Receptor (TLR) 2 and CD36/scavenger receptor-B2 (SR-B2), we studied how CD36/SR-B2 and TLR pathways promote Pg-mediated atherosclerosis. Western diet fed low density lipoprotein receptor knockout (Ldlr°) mice infected orally with Pg had a significant increase in lesion burden compared with uninfected controls. This increase was entirely CD36/SR-B2-dependent, as there was no significant change in lesion burden between infected and uninfected Ldlr° mice. Western diet feeding promoted enhanced CD36/SR-B2-dependent IL1β generation and foam cell formation as a result of Pg lipopolysaccharide (PgLPS) exposure. CD36/SR-B2 and TLR2 were necessary for inflammasome activation and optimal IL1ß generation, but also resulted in LPS induced lethality (pyroptosis). Modified forms of LDL inhibited Pg-mediated IL1ß generation in a CD36/SR-B2-dependent manner and prevented pyroptosis, but promoted foam cell formation. Our data show that Pg infection in the oral cavity can lead to significant TLR2-CD36/SR-B2 dependent IL1ß release. In the vessel wall, macrophages encountering systemic release of IL1ß, PgLPS and modified LDL have increased lipid uptake, foam cell formation, and release of IL1ß, but because pyroptosis is inhibited, this enables macrophage survival and promotes increased plaque development. These studies may explain increased lesion burden as a result of periodontal disease, and suggest strategies for development of therapeutics.     

The Microbiota Is Essential for the Generation of Black Tea Theaflavins-Derived Metabolites

Background

Theaflavins including theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3′-gallate (TF3′G), and theaflavin-3,3′-digallate (TFDG), are the most important bioactive polyphenols in black tea. Because of their poor systemic bioavailability, it is still unclear how these compounds can exert their biological functions. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans.

Methods and Findings

In the present study, we gavaged specific pathogen free (SPF) mice and germ free (GF) mice with 200 mg/kg TFDG and identified TF, TF3G, TF3′G, and gallic acid as the major fecal metabolites of TFDG in SPF mice. These metabolites were absent in TFDG- gavaged GF mice. The microbial bioconversion of TFDG, TF3G, and TF3′G was also investigated in vitro using fecal slurries collected from three healthy human subjects. Our results indicate that TFDG is metabolized to TF, TF3G, TF3′G, gallic acid, and pyrogallol by human microbiota. Moreover, both TF3G and TF3′G are metabolized to TF, gallic acid, and pyrogallol by human microbiota. Importantly, we observed interindividual differences on the metabolism rate of gallic acid to pyrogallol among the three human subjects. In addition, we demonstrated that Lactobacillus plantarum 299v and Bacillus subtilis have the capacity to metabolize TFDG.

Conclusions

The microbiota is important for the metabolism of theaflavins in both mice and humans. The in vivo functional impact of microbiota-generated theaflavins-derived metabolites is worthwhile of further study.     

Diazinon-induced ulcerative keratitis in C57bl/6 mice

As a well-known organophosphate insecticide, diazinon (DZN) has been used for several decades in agriculture. The major signs of ophthalmic toxicity of DZN have been reported to be cholinergic overstimulation (lacrimation, myosis). Here, we report, for the first time, ulcerative keratitis in C57bl/6 mice secondary to sub-acute exposure to DZN. Four groups of female C57bl/6 mice were administered intraperitoneally either DZN (1, 5, 25 mg/kg/day) or vehicle for 14 consecutive days. Then, histopathological examinations on mice eyes were performed using light microscope and scored for corneal keratitis. Furthermore, blood cholinesterase activity, and hematologic examinations were performed. Data indicated a significant ulcerative keratitis with prompt vision loss in mice exposed to 25 and 5 mg/kg/day (P < 0.05) doses. These results suggest that diazinon might induce ulcerative keratitis secondary to its immunosuppresive effects at high doses in C57bl/6 mice.     

Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.