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Background The etiology of food allergy is poorly understood; mouse models are powerful systems to discover immunologic pathways driving allergic disease. C3H/HeJ mice are a widely used model for the study of peanut allergy because, unlike C57BL/6 or BALB/c mice, they are highly susceptible to oral anaphylaxis. However, the immunologic mechanism of this strain’s susceptibility is not known. Objective We aimed to determine the mechanism underlying the unique susceptibility to anaphylaxis in C3H/HeJ mice. We tested the role of deleterious Toll-like receptor 4 (Tlr4) or dedicator of cytokinesis 8 (Dock8) mutations in this strain because both genes have been associated with food allergy. Methods We generated C3H/HeJ mice with corrected Dock8 or Tlr4 alleles and sensitized and challenged them with peanut. We then characterized the antibody response to sensitization, anaphylaxis response to both oral and systemic peanut challenge, gut microbiome, and biomarkers of gut permeability. Results In contrast to C3H/HeJ mice, C57BL/6 mice were resistant to anaphylaxis after oral peanut challenge; however, both strains undergo anaphylaxis with intraperitoneal challenge. Restoring Tlr4 or Dock8 function in C3H/HeJ mice did not protect from anaphylaxis. Instead, we discovered enhanced gut permeability resulting in ingested allergens in the bloodstream in C3H/HeJ mice compared to C57BL/6 mice, which correlated with an increased number of goblet cells in the small intestine. Conclusions Our work highlights the potential importance of gut permeability in driving anaphylaxis to ingested food allergens; it also indicates that genetic loci outside of Tlr4 and Dock8 are responsible for the oral anaphylactic susceptibility of C3H/HeJ mice.

Chronic rejection is among the most pressing clinical challenges in solid organ transplantation. Interestingly, in a mouse model of heterotopic heart transplantation, antibody-dependent, natural killer (NK) cell-mediated chronic cardiac allograft vasculopathy occurs in some donor-recipient strain combinations, but not others. In this study, we sought to identify the mechanism underlying this unexplained phenomenon. Cardiac allografts from major histocompatibility complex (MHC) mismatched donors were transplanted into immune-deficient C57Bl/6.rag-/- recipients, followed by administration of a monoclonal antibody against the donor MHC class I antigen. We found marked allograft vasculopathy in hearts from C3H donors, but near-complete protection of BALB/c allografts from injury. We found no difference in recipient NK cell phenotype or intrinsic responsiveness to activating signals between recipients of C3H versus BALB/c allografts. However, cardiac endothelial cells from C3H allografts showed an approximately twofold higher expression of Rae-1, an activating ligand of the NK cell receptor natural killer group 2D (NKG2D). Importantly, the administration of a neutralizing antibody against NKG2D abrogated the development of allograft vasculopathy in recipients of C3H allografts, even in the presence of donor-specific antibodies. Therefore, the activating NK cell receptor NKG2D is necessary in this model of chronic cardiac allograft vasculopathy, and strain-dependent expression of NK activating ligands correlates with the development of this disease.

As the largest organ exposed to the outside of mammals, skin has direct photosensitivity. Recent studies have even shown that cutaneous irradiation played a role in local circadian systems. However, whether it can further affect the central clock system is controversial. Here, plasm melatonin rhythm of melatonin-proficient C3H/He mice was assessed, and on this basis, a well-designed segmented lighting method was used to investigate the effects of dorsal skin irradiation on locomotor activity and plasm melatonin content in male C3H/He mice. In brief, mice were separately exposed to cutaneous irradiation, intraocular irradiation or darkness for 60 min at specific moments. The results showed that neither blue nor red cutaneous exposure had obvious effect on central rhythm oscillation while intraocular irradiation could significantly change the central clock of mice, and the effect of blue light was more forceful than red light. It suggests that intraocular nonvisual channels still play a dominant role in rhythmic regulation, which has not been challenged by the discovery of local light entrainment in exposed peripheral tissues.

The space radiation environment consists of multiple species of charged particles, including 28Si ions, that may impact brain function during and following missions. To develop biomarkers of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with 28Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following 28Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation could be used for the development of biomarker of the space radiation response.

Background Alopecia areata (AA) is an autoimmune disease resulting in non-scarring hair loss. Animal models are useful means to identify candidates for therapeutic agents. The C3H/HeJ mouse AA model induced via transferring cultured lymphoid cells isolated from AA-affected mice is widely used for AA research. However, this conventional method requires the continuous breeding of AA mice. Objective We aimed to establish a new method to generate AA model using the transfer of cryopreserved cells, which allows the rapid induction of a large number of AA mice when needed. Methods We cryopreserved lymph node cells soon after isolation from AA-affected mice and injected thawed-cultured cells into recipient mice. H&E staining, immunohistochemical staining, quantitative real-time PCR and ELISA were conducted to identify pathological characteristics. Flow cytometry was performed to reveal the profile of transferred cells. Results More than 90 % of recipient mice developed AA-like hair loss and showed inflammatory cell infiltration around anagen hair follicles, markedly increased mRNA expressions of interferon-γ, CXCL11, and granzyme B, and elevated interferon-α protein levels in the skin compared with naive mice. Higher percentages of effector memory T cells and dendritic cells in transferred cells resulted in a higher incidence of AA. Conclusion This is the first report to establish a method for generating AA mice using cryopreserved lymphocytes. These AA mice have similar pathological characteristics to AA mice generated with the conventional method and AA patients. This convenient and reproducible method is expected to be valuable for AA study.

The enteric nervous system, which regulates many gastrointestinal functions, is derived from neural crest cells (NCCs). Defective NCC migration during embryonic development may lead to enteric neuropathies such as Hirschsprung's disease (hindgut aganglionosis). Sox10 is known to be essential for cell migration but downstream molecular events regulating early NCC migration have not been fully elucidated. This study aimed to determine how Sox10 regulates migration of sacral NCCs toward the hindgut using Dominant megacolon mice, an animal model of Hirschsprung's disease with a Sox10 mutation.We used the following: time-lapse live cell imaging to determine the migration defects of mutant sacral NCCs; genome-wide microarrays, site-directed mutagenesis, and whole embryo culture to identify Sox10 targets; and liquid chromatography and tandem mass spectrometry to ascertain downstream effectors of Sox10.Sacral NCCs exhibited retarded migration to the distal hindgut in Sox10-null embryos with simultaneous down-regulated expression of cadherin-19 (Cdh19). Sox10 was found to bind directly to the Cdh19 promoter. Cdh19 knockdown resulted in retarded sacral NCC migration in vitro and ex vivo, whereas re-expression of Cdh19 partially rescued the retarded migration of mutant sacral NCCs in vitro. Cdh19 formed cadherin-catenin complexes, which then bound to filamentous actin of the cytoskeleton during cell migration.Cdh19 is a direct target of Sox10 during early sacral NCC migration toward the hindgut and forms cadherin-catenin complexes which interact with the cytoskeleton in migrating cells. Elucidation of this novel molecular pathway helps to provide insights into the pathogenesis of enteric nervous system developmental defects.

Misfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01191-w.

Nerves in bone play well‐established roles in pain and vasoregulation and have been associated with progression of skeletal disorders, including osteoporosis, fracture, arthritis, and tumor metastasis. However, isolation of the region‐specific mechanisms underlying these relationships is limited by our lack of quantitative methods for neuroskeletal analysis and precise maps of skeletal innervation. To overcome these limitations, we developed an optimized workflow for imaging and quantitative analysis of axons in and around the bone, including validation of Baf53b‐Cre in concert with R26R‐tdTomato (Ai9) as a robust pan‐neuronal reporter system for use in musculoskeletal tissues. In addition, we created comprehensive maps of sympathetic adrenergic and sensory peptidergic axons within and around the full length of the femur and tibia in two strains of mice (B6 and C3H). In the periosteum, these maps were related to the surrounding musculature, including entheses and myotendinous attachments to bone. Three distinct patterns of periosteal innervation (termed type I, II, III) were defined at sites that are important for bone pain, bone repair, and skeletal homeostasis. For the first time, our results establish a gradient of bone marrow axon density that increases from proximal to distal along the length of the tibia and define key regions of interest for neuroskeletal studies. Lastly, this information was related to major nerve branches and local maps of specialized mechanoreceptors. This detailed mapping and contextualization of the axonal subtypes innervating the skeleton is intended to serve as a guide during the design, implementation, and interpretation of future neuroskeletal studies and was compiled as a resource for the field as part of the NIH SPARC consortium. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Gastric cancer (GC) is a highly prevalent malignancy featured by dismal oncological outcomes. Accumulating pieces of evidence have consensus over the therapeutic significance of extracellular vesicles (EVs) and its role in carcinogenesis. Here, we planned to uncover EVs' role in GC by shuttling microRNA-1290 (miR-1290) and to identify the possible molecular mechanism associated with Grhl2, PD-L1, and ZEB1. Grhl2 was under-expressed in GC tissues, exhibiting a negative correlation with PD-L1 expression. In addition, Grhl2 promoted T cell proliferation by down-regulating PD-L1 via inhibiting ZEB1, while miR-1290 was found to negatively regulate Grhl2. EVs were also isolated from GC cells or normal gastric epithelial cells and identified with the presence of EV markers. miR-1290 expression was determined to be enriched in the EVs derived from GC cells and observed to promote the suppressive action of GC cells on T cell activation by up-regulating PD-L1 via the Grhl2/ZEB1 pathway in the co-culture system of GC cells with or without treatment of EVs with T cells. Moreover, we also developed a mouse model of GC and injected the EVs derived from miR-1290-inhibitor-treated GC cells into the tumor-bearing mice for further validation of mechanism in vivo. Intriguingly, the pivotal role of EVs-shuttled miR-1290 as an oncomiR was demonstrated in vivo. Collectively, we found that miR-1290 in EVs secreted from GC cells contributed to immune escape through the Grhl2/ZEB1/PD-L1 axis.

Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines, as such a vaccine has been proven to be successful in the past. The expression of recombinant antigens in meningococcal Outer Membrane Vesicles (OMVs), with the OMV functioning both as adjuvant and delivery vehicle, greatly enhances their potential. Immunization studies in mice have shown that OMV-based vaccines can protect against various pathogens and an OMV-based meningococcal vaccine is approved and available for human use. Because of its surface localization in Borrelia and the detailed knowledge regarding its immunogenicity and structure, OspA was chosen as a suitable lipoprotein to be tested as an OMV-based vaccine against Lyme borreliosis. We have previously shown that the OMV-OspA vaccine was immunogenic in mice and here we assessed the efficacy of OMV-OspA. We generated a second-generation OMV-OspA vaccine and vaccinated C3H/HeN mice with (EDTA extracted) meningococcal OMVs expressing OspA from B. burgdorferi strain B31. The adjuvant effect of empty OMVs on recombinant OspA was tested as well. We subsequently challenged mice with a subcutaneous injection of B. burgdorferi. Average antibody end-point titers against the OspA-OMV construct were high, although lower compared to the antibodies raised against recombinant OspA. Interestingly, antibody titers between recombinant OspA adjuvanted with aluminum hydroxide and recombinant OspA with OMV as adjuvant were comparable. Finally, qPCR and culture data show that both the OspA-OMV and the vaccine based on recombinant OspA with OMV as adjuvant provided significant, yet partial protection, against Borrelia infection. OMV-based vaccines using Borrelia (lipo)proteins are an easy and feasible vaccination method protecting against B. burgdorferi infection and could be a promising strategy in humans.

Immunotherapy and targeted therapy are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of immunotherapy (IT; anti-PD1)->targeted therapy (TT; ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS- (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT->TT. Tumor volumes were measured and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-Seq), and reverse phase protein analysis (RPPA). scRNA-Seq demonstrated the IT->TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells (DCs) and natural killer (NK) cells, and decreased numbers of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDCSs), and regulatory T cells (Tregs). Durable responses to the IT->TT sequence were dependent on T-cell activity, with depletion of CD8(+), but not CD4(+) T cells, abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT->TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT->TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront immunotherapy improves the responses to targeted therapy in BRAF- and NRAS-mutant melanoma models.

SummaryFrancisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell envelope-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG – a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1β and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1β and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1β, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

The hereditary disease Retinitis pigmentosa results in severe vision loss due to photoreceptor degeneration by unclear mechanisms. In several disease models, the second messenger cGMP accumulates in the degenerating photoreceptors, where it may over‐activate specific cGMP‐interacting proteins, like cGMP‐dependent protein kinase. Moreover, interventions that counteract the activity of these proteins lead to reduced photoreceptor cell death. Yet there is little or no information whether other than such regular cGMP‐interactors are present in the retina, which we, therefore, investigated in wild‐type and retinal degeneration (rd1, rd10, and rd2) mouse models. An affinity chromatography based proteomics approach that utilized immobilized cGMP analogs was applied to enrich and select for regular and potentially new cGMP‐interacting proteins as identified by mass spectrometry. This approach revealed 12 regular and 10 potentially new retinal cGMP‐interacting proteins (e.g., EPAC2 and CaMKIIα). Several of the latter were found to be expressed in the photoreceptors and to have proximity to cGMP and may thus be of interest when defining prospective therapeutic targets or biomarkers for retinal degeneration., The involvement of cGMP (cyclic guanosyl monophosphate) in the retinal degeneration mechanism(s) is elusive, so we applied a proteomics approach to look for cGMP‐binding proteins in healthy and degenerating retinas. This approach revealed known binding proteins (PKG, PKAs, and PDEs) as well as potential new cGMP‐interacting proteins (EPAC2 (RAPGEF4), p‐CaMKIIα, p‐GSK3β, and MAPK1/3). The potential new cGMP‐interacting proteins showed proximity with cGMP in situ in the retina. While an indirect or direct binding of cGMP to these proteins is not yet verified, the results indicate them as important proteins to decipher the cGMP signaling pathway and hence the degeneration mechanisms. Illustration created with https://biorender.com/.

16 p.-7 fig., Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of high acuity and color vision and, ultimately to blindness. In RP, a vast number of mutations perturb the structure and function of rod photoreceptors, while cones remain initially unaffected. Extensive rod loss in advanced stages of the disease triggers cone death by a mechanism that is still largely unknown. Here, we show that secondary cone cell death in animal models for RP is associated with increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at late stages of the disease, when the majority of rods have already degenerated, was sufficient to delay cone death and support long-term cone survival in two mouse models for RP, affected by mutations in the phosphodiesterase 6b gene. Moreover, the surviving cones remained light-sensitive, leading to an improvement in visual function. RNA-seq analysis of protected cones demonstrated that HDAC inhibition initiated multi-level protection via regulation of different pro-survival pathways,including MAPK, PI3K-Akt, and autophagy. This study suggests a unique opportunity for targeted pharmacological protection of secondary dying cones by HDAC inhibition and creates hope to maintain vision in RP patients even in advanced disease stages., This work was supported by the ProRetina foundation,the Kerstan Foundation, Deutsche Forschungsgemeinschaft (DFGTR 1238/4-1), Swiss National Science Foundation (31003A_173008),BMBF (FKZ: 01EK1613E), and GC2018-098557-B-I00 from MCIU/AEI/FEDER, UE. RGS is a recipient of a JIN grant RTI2018-098990- J-I00 from MCIU, Spain.

BACKGROUND: In children, the acute pyelonephritis that can result from urinary tract infections (UTIs), which commonly ascend from the bladder to the kidney, is a growing concern because it poses a risk of renal scarring and irreversible loss of kidney function. To date, the cellular mechanisms underlying acute pyelonephritis–driven renal scarring remain unknown. METHODS: We used a preclinical model of uropathogenic Escherichia coli–induced acute pyelonephritis to determine the contribution of neutrophils and monocytes to resolution of the condition and the subsequent development of kidney fibrosis. We used cell-specific monoclonal antibodies to eliminate neutrophils, monocytes, or both. Bacterial ascent and the cell dynamics of phagocytic cells were assessed by biophotonic imaging and flow cytometry, respectively. We used quantitative RT-PCR and histopathologic analyses to evaluate inflammation and renal scarring. RESULTS: We found that neutrophils are critical to control bacterial ascent, which is in line with previous studies suggesting a protective role for neutrophils during a UTI, whereas monocyte-derived macrophages orchestrate a strong, but ineffective, inflammatory response against uropathogenic, E. coli–induced, acute pyelonephritis. Experimental neutropenia during acute pyelonephritis resulted in a compensatory increase in the number of monocytes and heightened macrophage-dependent inflammation in the kidney. Exacerbated macrophage-mediated inflammatory responses promoted renal scarring and compromised renal function, as indicated by elevated serum creatinine, BUN, and potassium. CONCLUSIONS: These findings reveal a previously unappreciated outcome for neutrophil-macrophage imbalance in promoting host susceptibility to acute pyelonephritis and the development of permanent renal damage. This suggests targeting dysregulated macrophage responses might be a therapeutic tool to prevent renal scarring during acute pyelonephritis.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1β, from these cells in AA-affected skin.

Despite many hypothesized benefits of dietary isoflavone genistein (GEN) deriving from soy-based products, questions surrounding GEN’s developmental effects are increasing. To understand if in utero GEN exposure modulated postnatal respiratory allergies in the middle age, we conducted a time course study in the B6C3F1 offspring (PND 240–330) using a common household allergen (house dust mites: HDM; 10 μg/mouse for PND 240 and 290, and 50 μg/mouse for PND 330, a middle age in mice) following intranasal instillation, a physiological route of allergen exposure. GEN was administered to dams by gavage from gestational day 14 to parturition at a physiologically relevant dose (20 mg/kg body weight). Female and male offspring were sensitized with HDM allergens beginning about one month prior to sacrifice followed by challenges with three weekly dosings of HDM extracts, and they were euthanized at day 3 following the final HDM exposure. In utero exposure to GEN decreased HDM allergen-induced respiratory allergy in male B6C3F1 offspring at PND 330 as reflected by decreases in airway hyperresponsiveness (e.g., Penh value), HDM-specific IgG(1) (a Th2 type Ab) and the activity of eosinophil peroxidase in the lung (an indication of eosinophil recruitment to the lungs). However, in utero exposure to GEN had minimal effects on HDM allergen-induced respiratory allergy in the middle-aged female offspring. Changes in serum total IgE, HDM-specific IgE, and lung histopathology scores in both male and female offspring were not biologically significant. Overall, in utero GEN exposure exerted a protective effect on respiratory allergy in the middle-aged male, but not female, B6C3F1 offspring following later-life HDM exposures.

In this study we undertook a novel combination therapy using rAd-p53 in situ gene therapy and immunotherapy with immune checkpoint inhibitor (ICI) anti-PD-1 antibody for urogenital cancers. Three mouse syngeneic tumor cell lines, TRAMP-C2 (prostate cancer derived from C57BL/6 mice), MBT-2 (bladder cancer derived from C3H mice) and Renca (kidney cancer derived from BALB/c mice) were used in this study. The highest coxsackie and adenovirus receptor (CAR) mRNA expression was observed in TRAMP-C2 cells, followed by Renca and then MBT-2 cells. Consistent with the CAR expressions, rAd-p53 at 160 multiplicity of infection (MOI) significantly inhibited the cell proliferation of TRAMP-C2 and Renca cells, but not MBT-2 cells. In in vivo experiments, the combination of intratumoral injections of rAd-p53 (1 × 109 plaque-forming units) every other day and intraperitoneal injections of anti-mouse PD-1 antibody (200 μg) twice a week suppressed tumor growth and prolonged survival compared to rAd-p53 or anti-PD-1 antibody monotherapy in both the TRAMP-C2 and Renca models. Our results encourage the clinical development of combination therapy comprised of in situ gene therapy with rAd-p53 and immunotherapy with an ICI anti-PD-1 antibody for urogenital cancers.

Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for β1 and β3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to β1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.

Objective: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. Conclusions: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

Photoperiod or the duration of daylight has been implicated as a risk factor in the development of mood disorders. The dopamine and serotonin systems are impacted by photoperiod and are consistently associated with affective disorders. Hence, we evaluated, at multiple stages of postnatal development, the expression of key dopaminergic (TH) and serotonergic (Tph2, SERT, and Pet-1) genes, and midbrain monoamine content in mice raised under control Equinox (LD 12:12), Short winter-like (LD 8:16), or Long summer-like (LD 16:8) photoperiods. Focusing in early adulthood, we evaluated the midbrain levels of these serotonergic genes, and also assayed these gene levels in the dorsal raphe nucleus (DRN) with RNAScope. Mice that developed under Short photoperiods demonstrated elevated midbrain TH expression levels, specifically during perinatal development compared to mice raised under Long photoperiods, and significantly decreased serotonin and dopamine content throughout the course of development. In adulthood, Long photoperiod mice demonstrated decreased midbrain Tph2 and SERT expression levels and reduced Tph2 levels in the DRN compared Short photoperiod mice. Thus, evaluating gene × environment interactions in the dopaminergic and serotonergic systems during multiple stages of development may lead to novel insights into the underlying mechanisms in the development of affective disorders.

Background: Transthyretin (TTR) is a tetrameric, amyloid-β (Aβ)-binding protein, which reduces Aβ toxicity. The TTR/Aβ interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer’s disease. Objective: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aβ interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer’s disease using positron emission tomography (PET). Methods: Female mice (AβPPswe/PS1A246E/TTR+/–) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aβ in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age = 14 months. Results: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months. Conclusion: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions.

Background: Early-onset familial Alzheimer disease (EOFAD) is caused by heterozygous variants in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and APP genes. Decades after their discovery, the mechanisms by which these genes cause Alzheimer’s disease (AD) or promote AD progression are not fully understood. While it is established that presenilin (PS) enzymatic activity produces amyloid-β (Aβ), PSs also regulate numerous other cellular functions, some of which intersect with known pathogenic drivers of neurodegeneration. Accumulating evidence suggests that microglia, resident innate immune cells in the central nervous system, play a key role in AD neurodegeneration. Objective: Previous work has identified a regulatory role for PS2 in microglia. We hypothesized that PSEN2 variants lead to dysregulated microglia, which could further contribute to disease acceleration. To mimic the genotype of EOFAD patients, we created a transgenic mouse expressing PSEN2 N141I on a mouse background expressing one wildtype PS2 and two PS1 alleles. Results: Microglial expression of PSEN2 N141I resulted in impaired γ-secretase activity as well as exaggerated inflammatory cytokine release, NFκB activity, and Aβ internalization. In vivo, PS2 N141I mice showed enhanced IL-6 and TREM2 expression in brain as well as reduced branch number and length, an indication of “activated” morphology, in the absence of inflammatory stimuli. LPS intraperitoneal injection resulted in higher inflammatory gene expression in PS2 N141I mouse brain relative to controls. Conclusion: Our findings demonstrate that PSEN2 N141I heterozygosity is associated with disrupted innate immune homeostasis, suggesting EOFAD variants may promote disease progression through non-neuronal cells beyond canonical dysregulated Aβ production.

For over a century, inbred mice have been used in many areas of genetics research to gain insight into the genetic variation underlying traits of interest. The generalizability of any genetic research study in inbred mice is dependent upon all individual mice being genetically identical, which in turn is dependent on the breeding designs of companies that supply inbred mice to researchers. Here, we compare whole-genome sequences from individuals of four commonly used inbred strains that were procured from either the colony nucleus or from a production colony (which can be as many as ten generations removed from the nucleus) of a large commercial breeder, in order to investigate the extent and nature of genetic variation within and between individuals. We found that individuals within strains are not isogenic, and there are differences in the levels of genetic variation that are explained by differences in the genetic distance from the colony nucleus. In addition, we employ a novel approach to mutation rate estimation based on the observed genetic variation and the expected site frequency spectrum at equilibrium, given a fully inbred breeding design. We find that it provides a reasonable per nucleotide mutation rate estimate when mice come from the colony nucleus (~7.9 × 10−9 in C3H/HeN), but substantially inflated estimates when mice come from production colonies.

BACKGROUND The putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent. QUESTIONS/PURPOSES (1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma. METHODS In this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H2O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation. RESULTS Constitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm versus 1316 ± 387.4 mm, mean difference 1009 mm [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm versus 326.1 ± 72.8 mm, mean difference 326.1 mm [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10 ± 8.3x10 photons/s versus 9.3 x 10 ± 1.5 x 10 photons/s, mean difference 8.6 x 10 photons/s [95% CI 5.1 x 10 to 1.2 x 10]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm versus 1335 ± 102.7 mm, mean difference 840.3 mm [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions. CONCLUSIONS As demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted. CLINICAL RELEVANCE Our results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.

Cell cycle-related kinase (CCRK) has a conserved role in ciliogenesis, and Ccrk defects in mice lead to developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, retinal degeneration, and polycystic kidney. Here, we found that Ccrk is highly expressed in mouse trachea and bronchioles. Ccrk mutants exhibited pulmonary hypoplasia and abnormal branching morphogenesis in respiratory organ development. Furthermore, we demonstrated that Ccrk mutant lungs exhibit not only impaired branching morphogenesis but also a significant sacculation deficiency in alveoli associated with reduced epithelial progenitor cell proliferation. In pseudoglandular stages, Ccrk mutant lungs showed a downregulation of Hedgehog (Hh) signaling and defects in cilia morphology and frequency during progenitor-cell proliferation. Interestingly, we observed that activation of the Hh signaling pathway by small-molecule smoothened agonist (SAG) partially rescued bud morphology during branch bifurcation in explants from Ccrk mutant lungs. Therefore, CCRK properly regulates respiratory airway architecture in part through Hh-signal transduction and ciliogenesis. [BMB Reports 2020; 53(7): 367-372].

Background/aim We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression. Materials and methods We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia. The total RNA extracted from each brain's microglia was subjected to DNA microarray analysis. Results Many genes were found to be significantly differentially expressed between hard- and soft-diet-fed mice in each group of the same feeding period. The expression of several genes involved in the regulation of actin cytoskeleton was down-regulated in the soft-diet-fed mice. Conclusion Mastication may affect microglia's roles in cognition as well as their neuroimmune activity through their activity of patrolling the brain.

The aim of this study was to investigate the behavioral, immunological, and neurological effects of long-term isolation in an animal model. Male C3H/eB mice wereraised in either social isolation or...

Host-associated reservoirs account for the majority of recurrent and oftentimes recalcitrant infections. Previous studies established that uropathogenic E. coli – the primary cause of urinary tract infections (UTIs) – can adhere to vaginal epithelial cells preceding UTI. Here, we demonstrate that diverse urinary E. coli isolates not only adhere to, but also invade vaginal cells. Intracellular colonization of the vaginal epithelium is detected in acute and chronic murine UTI models indicating the ability of E. coli to reside in the vagina following UTI. Conversely, in a vaginal colonization model, E. coli are detected inside vaginal cells and the urinary tract, indicating that vaginal colonization can seed the bladder. More critically, bacteria are identified inside vaginal cells from clinical samples from women with a history of recurrent UTI. These findings suggest that E. coli can establish a vaginal intracellular reservoir, where it may reside safely from extracellular stressors prior to causing an ascending infection., Uropathogenic E. coli can adhere to vaginal epithelial cells preceding urinary tract infection (UTI). Here, Brannon et al. show that urinary E. coli isolates can not only adhere to, but also invade vaginal cells in mouse UTI models and in clinical samples obtained from women with recurrent UTI.

Microbiota, host and dietary metabolites/signals compose the rich gut chemical environment, which profoundly impacts virulence of enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) engages a syringe-like machinery named type-III secretion system (T3SS) to inject effectors within host cells that lead to intestinal colonization and disease. We previously conducted a high-throughput screen to identify metabolic pathways that affect T3SS expression. Here we show that in the presence of arginine, the arginine sensor ArgR, identified through this screen, directly activates expression of the genes encoding the T3SS. Exogenously added arginine induces EHEC virulence gene expression in vitro. Congruently, a mutant deficient in arginine transport (Δ artP ) had decreased virulence gene expression. ArgR also augments murine disease caused by Citrobacter rodentium , which is a murine pathogen extensively employed as a surrogate animal model for EHEC. The source of arginine sensed by C. rodentium is not dietary. At the peak of C. rodentium infection, increased arginine concentration in the colon correlated with down-regulation of the host SLC7A2 transporter. This increase in the concentration of colonic arginine promotes virulence gene expression in C. rodentium . Arginine is an important modulator of the host immune response to pathogens. Here we add that arginine also directly impacts bacterial virulence. These findings suggest that a delicate balance between host and pathogen responses to arginine occur during disease progression.

RaoN is a Salmonella-specific small RNA that is encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. We previously reported that RaoN is induced under conditions of acid and oxidative stress combined with nutrient limitation, contributing to the intramacrophage growth of Salmonella enterica serovar Typhimurium. However, the role of RaoN in nitrosative stress response and virulence has not yet been elucidated. Here we show that the raoN mutant strain has increased susceptibility to nitrosative stress by using a nitric oxide generating acidified nitrite. Extending previous research on the role of RaoN in oxidative stress resistance, we found that NADPH oxidase inhibition restores the growth of the raoN mutant in LPS-treated J774A.1 macrophages. Flow cytometry analysis further revealed that the inactivation of raoN leads to an increase in the intracellular level of reactive oxygen species (ROS) in Salmonella-infected macrophages, suggesting that RaoN is involved in the inhibition of NADPH oxidase-mediated ROS production by mechanisms not yet resolved. Moreover, we evaluated the effect of raoN mutation on the virulence in murine systemic infection and determined that the raoN mutant is less virulent than the wild-type strain following oral inoculation. In conclusion, small regulatory RNA RaoN controls nitrosative-oxidative stress resistance and is required for virulence of Salmonella in mice.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Dampened prepulse inhibition (PPI) is a consistent observation in psychiatric disorders, including schizophrenia and qualifies as a robust endophenotype for genetic evaluation. Using high PPI C57BL/6NCrlCrlj (B6Nj) and low PPI C3H/HeNCrlCrlj (C3HNj) inbred mouse strains, we have previously reported a quantitative trait locus (QTL) for PPI at chromosome 10 and identified Fabp7 as a candidate gene for regulating PPI and schizophrenia pathogenesis using Fabp7-deficient mice (B6.Cg-Fabp7 KO). Here, considering a possibility of carryover of residual genetic materials from embryonic stem (ES) cells used in generating knockout (KO) mice, we set out to re-address the genotype-phenotype correlation in a uniform genetic background. By generating a new Fabp7 KO mouse model in C57BL/6NCrl (B6N) background using the CRISPR-Cas9 nickase system, we evaluated the impact of Fabp7 ablation on schizophrenia-related behavioral phenotypes. To our surprise, we found no significant differences in PPI or any of the schizophrenia-related behavioral scores, as observed in our previous B6.Cg-Fabp7 KO mice. We identified several C3H/He mouse strain-specific alleles within the interval of chromosome 10-QTL, which are shared with 129/Sv mouse strains. These alleles, derived from 129/Sv ES cells, were retained in the B6.Cg-Fabp7 KO, despite multiple backcrossing and are thought to be responsible for the dampened PPI. In summary, our study demonstrates a precise genotype-phenotype relation for Fabp7 loss-of-function in a uniform B6N background, and raises the necessity of further analysis of the effects of genomic variants flanking the Fabp7 interval on phenotypes.

Sepsis is a major challenge in clinical practice and responsible for high mortality. Recent studies indicated that integrins participated in toll-like-receptor (TLR)-mediated innate immunity. In the present study, we investigated the mechanism of integrin β3 and TLR4 signaling using a cecal ligation and puncture (CLP)-induced sepsis and lipopolysaccharide (LPS)-treated macrophage cell model. In a lethal CLP model, the survival rate of integrin β3 mice was higher than that of wild-type mice. The levels of alanine aminotransferase, aspartate transaminase, creatinine, blood urea nitrogen , and lactate dehydrogenase in the serum and cluster of differentiation 14 (CD14) protein expression in the tissues were significantly decreased in integrin β3 mice. A similar effect with regard to CD14 down-regulation was observed in septic TLR4 mice. In wild-type macrophages, the inhibition of integrin β3 by P11 or with a specific antibody, inhibited TNF-α, and IL-6 release at the early time period of LPS stimulation. However, during the late periods of LPS stimulation this effect was not noted. CD14 expression levels had no change in such treatment. In contract, LPS-induced TNF-α and IL-6 release and LPS-induced CD14 expression were significantly decreased in integrin β3macrophages. The inhibition of the TLR4 pathway by TAK-242, or in TLR4 mutant macrophages abolished LPS-induced CD14 expression. Integrin β3 pathway activation by vitronectin exhibited no effect in CD14 expression. Furthermore, recombinant CD14 protein stimulation reversed integrin β3 deficiency and caused lower TNF-α and IL-6 release. Moreover, the molecular interaction of TLR4 and integrin β3 was significantly increased following LPS stimulation. In conclusion, integrin β3 positively regulated TLR4-mediated inflammatory responses via CD14 expression in macrophages in septic condition. Specifically targeting integrin β3/TLR4-CD14 signaling pathway may be a potential treatment strategy for polymicrobial sepsis.

The transition metal nickel is used in a wide variety of alloys and medical devices. Nickel can cause a range of toxicities from allergy in humans to tumors when implanted in animals. Several microarray studies have examined nickel toxicity, but so far none have comprehensively profiled expression over an extended period. In this work, male mice were implanted with a single nickel pellet in the muscle of the right leg with the left leg used as a control. At 3 week intervals up to 12 months, nickel concentrations in bioflulids and microarrays of surrounding tissue were used to track gene expression patterns. Pellet biocorrosion resulted in varying levels of systemic nickel over time, with peaks of 600 μg L−1 in serum, while global gene expression was cyclical in nature with immune related genes topping the list of overexpressed genes. IPA and KEGG pathway analyses was used to attribute overall biological function to changes in gene expression levels, supported by GO enrichment analysis. IPA pathways identified sirtuin, mitochondria, and oxidative phosphorylation as top pathways, based predominantly on downregulated genes, whereas immune processes were associated with upregulated genes. Top KEGG pathways identified were lysosome, osteoclast differentiation, and phasgosome. Both pathway approaches identified common immune responses, as well as hypoxia, toll like receptor, and matrix metalloproteinases. Overall, pathway analysis identified a negative impact on energy metabolism, and a positive impact on immune function, in particular the acute phase response. Inside the cell the impacts were on mitochondria and lysosome. New pathways and genes responsive to nickel were identified from the large dataset in this study which represents the first long-term analysis of the effects of chronic nickel exposure on global gene expression.

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Prolonged endoplasmic reticulum stress has been identified in various diseases. Inflammatory mediators, which have been shown to induce endoplasmic reticulum stress in several studies, have been suggested to serve as the important modulators in pain development. In this study, the authors hypothesized that the endoplasmic reticulum stress triggered by inflammatory mediators contributed to pain development. Methods The authors used a male mouse model of bone cancer pain. The control mice were intrathecally injected with tumor necrosis factor-α (TNF-α) and lipopolysaccharide, the bone cancer pain mice were intrathecally injected with the endoplasmic reticulum stress inhibitors 4-PBA and GSK2606414. The nociceptive behaviors, endoplasmic reticulum stress markers, and inflammatory mediators were assessed. Results Increased expression of the p-RNA-dependent protein kinase-like endoplasmic reticulum kinase and p-eukaryotic initiation factor 2α were found in the spinal neurons during bone cancer pain, along with upregulation of inflammatory mediators (TNF-α, interleukin 1β, and interleukin 6). Intrathecal administration of TNF-α or lipopolysaccharide increased the expression of endoplasmic reticulum stress markers in control mice. Inhibition of endoplasmic reticulum stress by intrathecal administration of 4-PBA (baseline vs. 3 h: 0.34 ± 0.16 g vs. 1.65 ± 0.40 g in paw withdrawal mechanical threshold, 8.00 ± 1.20 times per 2 min vs. 0.88 ± 0.64 times per 2 min in number of spontaneous flinches, P < 0.001, n = 8) or GSK2606414 (baseline vs. 3 h: 0.37 ± 0.08 g vs. 1.38 ± 0.11 g in paw withdrawal mechanical threshold, 8.00 ± 0.93 times per 2 min vs. 3.25 ± 1.04 times per 2 min in number of spontaneous flinches, P < 0.001, n = 8) showed time- and dose-dependent antinociception. Meanwhile, decreased expression of inflammatory mediators (TNF-α, interleukin 1β, and interleukin 6), as well as decreased activation of astrocytes in the spinal cord, were found after 4-PBA or GSK2606414 treatment. Conclusions Inhibition of inflammatory mediator–triggered endoplasmic reticulum stress in spinal neurons attenuates bone cancer pain via modulation of neuroinflammation, which suggests new approaches to pain relief.

Chronic allograft rejection is the most common cause of long-term allograft failure. One reason is that current diagnostics and therapeutics for chronic allograft rejection are very limited. We here show that enhanced NFκB signaling in kidney grafts contributes to chronic active antibody-mediated rejection (CAAMR), which is a major pathology of chronic kidney allograft rejections. Moreover, we found that urinary orosomucoid 1 (ORM1) is a candidate marker molecule and therapeutic target for CAAMR. Indeed, urinary ORM1 concentration was significantly higher in kidney transplant recipients pathologically diagnosed with CAAMR than in kidney transplant recipients with normal histology, calcineurin inhibitor toxicity, or interstitial fibrosis and tubular atrophy. Additionally, we found that kidney biopsy samples with CAAMR expressed more ORM1 and had higher NFκB and STAT3 activation in tubular cells than samples from non-CAAMR samples. Consistently, ORM1 production was induced after cytokine-mediated NFκB and STAT3 activation in primary kidney tubular cells. The loss- and gain-of-function of ORM1 suppressed and promoted NFκB activation, respectively. Finally, ORM1-enhanced NFκB-mediated inflammation development in vivo. These results suggest that an enhanced NFκB-dependent pathway following NFκB and STAT3 activation in the grafts is involved in the development of chronic allograft rejection after kidney transplantation and that ORM1 is a non-invasive candidate biomarker and possible therapeutic target for chronic kidney allograft rejection.

Rationale: Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production by hyper-activated B cells. Although mesenchymal stem cells (MSCs) ameliorate lupus symptoms by inhibiting T cells, whether they inhibit B cells has been controversial. Here we address this issue and reveal how to prime MSCs to inhibit B cells and improve the efficacy of MSCs in SLE. Methods: We examined the effect of MSCs on purified B cells in vitro and the therapeutic efficacy of MSCs in lupus-prone MRL.Faslpr mice. We screened chemicals for their ability to activate MSCs to inhibit B cells. Results: Mouse bone marrow-derived MSCs inhibited mouse B cells in a CXCL12-dependent manner, whereas human bone marrow-derived MSCs (hMSCs) did not inhibit human B (hB) cells. We used a chemical approach to overcome this hurdle and found that phorbol myristate acetate (PMA), phorbol 12,13-dibutyrate, and ingenol-3-angelate rendered hMSCs capable of inhibiting IgM production by hB cells. As to the mechanism, PMA-primed hMSCs attracted hB cells in a CXCL10-dependent manner and induced hB cell apoptosis in a PD-L1-dependent manner. Finally, we showed that PMA-primed hMSCs were better than naive hMSCs at ameliorating SLE progression in MRL.Faslpr mice. Conclusion: Taken together, our data demonstrate that phorbol esters might be good tool compounds to activate MSCs to inhibit B cells and suggest that our chemical approach might allow for improvements in the therapeutic efficacy of hMSCs in SLE.

International audience; Gliadins are major wheat allergens. Their treatment by acid or enzymatic hydrolysis has been shown to modify their allergenic potential. As the interaction of food proteins with dendritic cells (DCs) is a key event in allergic sensitization, we wished to investigate whether deamidation and enzymatic hydrolysis influence gliadin processing by DC and to examine the capacity of gliadins to activate DCs. We compared the uptake and degradation of native and modified gliadins by DCs using mouse bone marrow-derived DCs. We also analyzed the effects of these interactions on the phenotypes of DCs and T helper (Th) lymphocytes. Modifying gliadins induced a change in physicochemical properties (molecular weight, hydrophobicity, and sequence) and also in the peptide size. These alterations in turn led to increased uptake and intracellular degradation of the proteins by DCs. Native gliadins (NGs) (100 μg/mL), but not modified gliadins, increased the frequency of DC expressing CD80 (15.41 ± 2.36% vs 6.81 ± 1.10%, p < 0.001), CCR7 (28.53 ± 8.17% vs 17.88 ± 2.53%, p < 0.001), CXCR4 (70.14 ± 4.63% vs 42.82 ± 1.96%, p < 0.001), and CCR7-dependent migration (2.46 ± 1.45 vs 1.00 ± 0.22, p < 0.01) compared with NGs. This was accompanied by Th lymphocyte activation (30.37 ± 3.87% vs 21.53 ± 3.14%, p < 0.1) and proliferation (16.39 ± 3.97% vs 9.31 ± 2.80%, p > 0.1). Moreover, hydrolysis decreases the peptide size and induces an increase in gliadin uptake and degradation. Deamidation and extensive enzymatic hydrolysis of gliadins modify their interaction with DCs, leading to alteration of their immunostimulatory capacity. These findings demonstrate the strong relationship between the biochemical characteristics of proteins and immune cell interactions.

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract, but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences between mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of SNPs and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche.IMPORTANCEUnderstanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intra-species variation as well as host anti-microbial defense mechanisms in defining commensal interactions.

BB0405 is a surface exposed Borrelia burgdorferi protein and its vaccination protected mice against B. burgdorferi infection. As BB0405 is highly conserved across different B. burgdorferi sensu lato species, we investigated whether vaccination with recombinant BB0405 or through intradermal bb0405 DNA tattoo vaccination could provide protection against different Borrelia species, specifically against Borrelia afzelii, the predominant B. burgdorferi sensu lato genospecies causing Lyme borreliosis across Eurasia. We immunized C3H/HeN mice with recombinant BB0405 or with a codon-optimized bb0405 DNA vaccine using the pVAC plasmid and immunized corresponding control groups mice with only adjuvant or empty vectors. We subsequently subjected these immunized mice to a tick challenge with B. afzelii CB43-infected Ixodes ricinus nymphs. Upon vaccination, recombinant BB0405 induced a high total IgG response, but bb0405 DNA vaccination did not elicit antibody responses. Both vaccine formulations did not provide protection against Borrelia afzelii strain CB43 after tick challenge. In an attempt to understand the lack of protection of the recombinant vaccine, we determined expression of BB0405 and showed that B. afzelii CB43 spirochetes significantly and drastically downregulate the expression of BB0405 protein at 37 °C compared to 33 °C, where as in B. burgdorferi B31 spirochetes expression levels remain unaltered. Vaccination with recombinant BB0405 was previously shown to protect against B. burgdorferi sensu stricto. Here we show that vaccination with either recombinant BB0405 (or non-immunogenic bb0405 DNA), despite being highly conserved among B. burgdorferi sl genospecies, does not provide cross-protection against B. afzelii, mostly likely due to downregulation of this protein in B. afzelii in the mammalian host.

Housing conditions can affect the well-being of laboratory animals and thereby affect the outcomes of experiments. The appropriate environment is essential for the expression of natural behavior in animals. Here, we compared survival rates in four inbred mouse strains maintained under three different environmental conditions. Three mouse strains (C57BL/6J, C3H/HeN, and DBA/2J) housed under environmental enrichment (EE) conditions showed improved survival; however, EE did not alter the survival rate of the fourth strain, BALB/c. None of the strains showed significant differences in body weights or plasma corticosterone levels in the three environmental conditions. For BALB/c mice, the rates of debility were higher in the EE group. Interestingly, for C57BL/6J and C3H/HeN mice, the incidence of animals with alopecia was significantly lower in the EE groups than in the control group. It is possible that the enriched environment provided greater opportunities for sheltering in a secure location in which to avoid interactions with other mice. The cloth mat flooring used for the EE group was bitten and chewed by the mice. Our findings suggest that depending on the mouse strains different responses to EE are caused with regard to health and survival rates. The results of this study provide basic data for further studies on EE.

Multiple drug discovery initiatives for tuberculosis are currently ongoing to identify and develop new potent drugs with novel targets in order to shorten treatment duration. One of the drug classes with a new mode of action is DprE1 inhibitors targeting an essential process in cell wall synthesis of Mycobacterium tuberculosis. In this investigation, three DprE1 inhibitors currently in clinical trials, TBA-7371, PBTZ169, and OPC-167832, were evaluated side-by-side as single agents in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon tuberculosis infection. The goal was to confirm the efficacy of the DprE1 inhibitors in a mouse tuberculosis model with advanced pulmonary pathology and perform comprehensive analysis of plasma, lung, and lesion-centric drug levels to establish pharmacokinetic-pharmacodynamic (PK-PD) parameters predicting efficacy at the site of infection. Results showed significant efficacy for all three DprE1 inhibitors in the C3HeB/FeJ mouse model after 2 months of treatment. Superior efficacy was observed for OPC-167832 even at low-dose levels, which can be attributed to its low MIC, favorable distribution, and sustained retention above the MIC throughout the dosing interval in caseous necrotic lesions, where the majority of bacteria reside in C3HeB/FeJ mice. These results support further progression of the three drug candidates through clinical development for tuberculosis treatment.

The metabolic syndrome (MetS), defined as the co-occurrence of disorders including obesity, dyslipidemia, insulin resistance, and hepatic steatosis, has become increasingly prevalent in the world over recent decades. Dietary and other environmental factors interacting with genetic predisposition are likely contributors to this epidemic. Among the involved dietary factors, excessive fructose consumption may be a key contributor. When fructose is consumed in large amounts, it can quickly produce many of the features of MetS both in humans and mice. The mechanisms by which fructose contributes to metabolic disease and its potential interactions with genetic factors in these processes remain uncertain. Here, we generated a small F2 genetic cohort of male mice derived from crossing fructose-sensitive and -resistant mouse strains to investigate the interrelationships between fructose-induced metabolic phenotypes and to identify hepatic transcriptional pathways that associate with these phenotypes. Our analysis indicates that the hepatic transcriptional pathways associated with fructose-induced hypertriglyceridemia and hyperinsulinemia are distinct from those that associate with fructose-mediated changes in body weight and liver triglyceride. These results suggest that multiple independent mechanisms and pathways may contribute to different aspects of fructose-induced metabolic disease.

BackgroundCombination therapy has been explored for advanced head and neck squamous cell carcinoma (HNSCC) owing to the limited efficacy of anti-epidermal growth factor receptor (EGFR) therapy. Increased expression and glycosylation of immune checkpoint molecules in tumors are responsible for cetuximab therapy refractoriness. The role of programmed death ligand 2 (PD-L2), a ligand of PD-1, in the immune function is unclear. Here, we examined the regulatory mechanism of PD-L2 glycosylation and its role in antitumor immunity and cetuximab therapy.MethodsSingle-cell RNA sequencing and immunohistochemical staining were used to investigate PD-L2 expression in cetuximab-resistant/sensitive HNSCC tissues. The mechanism of PD-L2 glycosylation regulation was explored in vitro. The effects of PD-L2 glycosylation on immune evasion and cetuximab efficacy were verified in vitro and using mice bearing orthotopic SCC7 tumors.ResultsThe PD-L2 levels were elevated and N-glycosylated in patients with cetuximab-resistant HNSCC. Glycosylated PD-L2 formed a complex with EGFR, which resulted in the activation of EGFR/signal transducer and activator of transcription 3 (STAT3) signaling and decreased the cetuximab binding affinity to EGFR. The N-glycosyltransferase fucosyltransferase (FUT8), a transcriptional target of STAT3, was required for PD-L2 glycosylation. Moreover, glycosylation modification stabilized PD-L2 by blocking ubiquitin-dependent lysosomal degradation, which consequently promoted its binding to PD-1 and immune evasion. Inhibition of PD-L2 glycosylation using Stattic, a specific STAT3 inhibitor, or PD-L2 mutation blocking its binding to FUT8, increased cytotoxic T lymphocyte activity and augmented response to cetuximab.ConclusionsIncreased expression and glycosylation of PD-L2 in tumors are an important mechanism for cetuximab therapy refractoriness. Thus, the combination of PD-L2 glycosylation inhibition and cetuximab is a potential therapeutic strategy for cancer.

Trpc7 (transient receptor potential cation channel, subfamily C, member 7; 862 amino acids) knockout mice are described showing no clear phenotypic alterations, therefore, the functional relevance of the gene remains unclear. A complementary approach for the functional analysis of a given gene is the examination of individuals harbouring a mutant allele of the gene. In the phenotype-driven Munich ENU mouse mutagenesis project, a high number of phenotypic parameters was used for establishing novel mouse models on the genetic background of C3H inbred mice. The phenotypically dominant mutant line SMA002 was established and further examined. Analysis of the causative mutation as well as the phenotypic characterization of the mutant line were carried out. The causative mutation was detected in the gene Trpc7 which leads to the production of a truncated protein due to the novel stop codon at amino acid position 810 thereby affecting the highly conserved cytoplasmic C terminus of the protein. Trpc7 heterozygous mutant mice of both sexes were viable and fertile, but showed distinct morphological and behavioural alterations which is in contrast to the published phenotype of Trpc7 knockout mice. Thus, the Trpc7K810Stop mutation leads to a dominant negative effect of the mutant protein.

Riboflavin is an essential micronutrient, but its transport and utilization have remained largely understudied among pathogenic spirochetes. Here, we show that Borrelia burgdorferi, the zoonotic spirochete that causes Lyme disease, is able to import riboflavin via products of its rfuABCD-like operon as well as synthesize flavin mononucleotide and flavin adenine dinucleotide despite lacking canonical genes for their synthesis. Additionally, a mutant deficient in the rfuABCD-like operon is resistant to the antimicrobial effect of roseoflavin, a natural riboflavin analog, and is attenuated in a murine model of Lyme borreliosis. Our combined results indicate not only that are riboflavin and the maintenance of flavin pools essential for B. burgdorferi growth but also that flavin utilization and its downstream products (e.g., flavoproteins) may play a more prominent role in B. burgdorferi pathogenesis than previously appreciated.