Establishment and visual analysis of CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice.

In CBA/J and C3H/HeJ mice, retinitis pigmentosa is inherited as an autosomal-recessive trait due to a mutation in Pde6b, which encodes cGMP phosphodiesterase subunit b. In these strains, the Y347X mutation in Pde6b leads to the upregulation of cGMP levels, increased Ca2+ influx induces rod death, and the outer segment and rod cells entirely disappeared by 35 days after birth. In the present study, we utilized the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9-mediated gene editing to repair the Y347X mutation in CBA/J and C3H/HeJ mice. Evaluation of the established CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice, which were confirmed to have normal retinal layers by live fundoscopic imaging and histopathological analysis, revealed improved visual acuity based on the visual cliff and light/dark latency tests. Furthermore, our analyses revealed that the visible platform test was a more effective tool for testing visual behavior in these mice. The results suggest that the established strains can serve as control groups for CBA/J and C3H/HeJ in ophthalmology studies involving retinitis pigmentosa.

Induction of food-specific IgG by Gene Gun-delivered DNA vaccines.

Background: Shellfish and tree nut allergies are among the most prevalent food allergies, now affecting 2%-3% and 1% of the US population, respectively. Currently, there are no approved therapies for shellfish or tree nut allergies, with strict avoidance being the standard of care. However, oral immunotherapy for peanut allergy and subcutaneous immunotherapy for environmental allergens are efficacious and lead to the production of allergen-specific IgG, which causes suppression of allergen effector cell degranulation. Since allergen-specific IgG is a desired response to alleviate IgE-mediated allergies, we tested transcutaneously-delivered DNA vaccines targeting shellfish and tree nut allergens for their ability to induce antigen-specific IgG, which would have therapeutic potential for food allergies. Methods: We assessed Gene Gun-delivered DNA vaccines targeting either crustacean shellfish or walnut/pecan allergens, with or without IL-12, in naïve mice. Three strains of mice, BALB/cJ, C3H/HeJ and CC027/GeniUnc, were evaluated for IgG production following vaccination. Vaccines were administered twice via Gene Gun, three weeks apart and then blood was collected three weeks following the final vaccination. Results: Vaccination with shellfish allergen DNA led to increased shrimp-specific IgG in all three strains, with the highest production in C3H/HeJ from the vaccine alone, whereas the vaccine with IL-12 led to the highest IgG production in BALB/cJ and CC027/GeniUnc mice. Similar IgG production was also induced against lobster and crab allergens. For walnut/pecan vaccines, BALB/cJ and C3H/HeJ mice produced significantly higher walnut- and pecan-specific IgG with the vaccine alone compared to the vaccine with IL-12, while the CC027 mice made significantly higher IgG with the addition of IL-12. Notably, intramuscular administration of the vaccines did not lead to increased antigen-specific IgG production, indicating that Gene Gun administration is a superior delivery modality. Conclusions: Overall, these data demonstrate the utility of DNA vaccines against two lifelong food allergies, shellfish and tree nuts, suggesting their potential as a food allergy therapy in the future.

Oral anaphylaxis to peanut in a mouse model is associated with gut permeability but not with Tlr4 or Dock8 mutations.

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.

Altered T cell subpopulations and serum anti-TYRP2 and tyrosinase antibodies in the acute and chronic phase of alopecia areata in the C3H/HeJ mouse model.

BACKGROUND: C3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear. OBJECTIVE: We aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model. METHODS: We analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer. RESULTS: The CD8+NKG2D+ T and CD8+ TEM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4+ TEM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8+ TEM/TRM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c+ cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8+ T cell percentages were upregulated in AA mice. CONCLUSION: These results suggest that the CD8+ and CD4+ T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model.

The antiinflammatory effects of Xuefu Zhuyu decoction on C3H/HeJ mice with alopecia areata.

BACKGROUND: As a traditional and typical prescription of prominently activating blood circulation to remove blood stasis, Xuefu Zhuyu decoction (XZD) consists of 15 kinds of herbal medicine. Clinical investigations have showed that XZD could significantly promote the new hair generation of alopecia areata (AA) patients characterized by Qi stagnation and blood stasis. PURPOSE: The purpose of this study was executed to determine whether the mechanisms by which XZD stimulated newborn hair were related to its anti-inflammatory effects. METHODS: Clinical AA individuals were recruited to confirm the efficies of XZD. High performance liquid chromatography (HPLC) analysis was performed to qualitatively and quantitatively determine the contents of 15 compounds in XZD. Schrodinger molecular docking and in vivo surface plasmon resonance (SPR) techniques were used to evaluate the potential binding properties of compounds to target proteins. C3H/HeJ mice were randomly assigned to groups control, AA, and the XZD administration (6.5, 13.0 and 26.0 g/kg/d). Except for mice in control group, all the mice in the other groups were treated with a 21-day chronic unpredictable mild stress (CUMS) induced AA. Hematoxylin-eosin (H&E) staining was performed to determine the degree of pathological damage to the skin. Enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) and in serum and skin tissues. Western blot, immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to examine the expression levels of IL-6, IL-1ß, TNF-α and osteopontin proteins and genes in skin tissues. RESULTS: XZD could visibly promote hair regeneration of AA patients. The potential active ingredients in XZD prescription included at least amygdalin, hydroxysafflor yellow A, kaempferide, ferulic acid, catalpol, verbascoside, ß-ecdysone, platycodin D, paeoniflorin, naringin, neohesperidin, liquiritin, glycyrrhizic acid, saikosaponin A and saikosaponin D. The results of molecular docking and SPR analysis showed that verbascoside, liquiritin, kaempferide and amygdalin showed the best potential binding properties with IL-6, IL-1ß, TNF-α and osteopontin, respectively. Pathological evaluation showed that compared with the CUMS group, the administration of XZD significantly promoted hair regeneration, evidenced by increased number of skin hair follicles in C3H/HeJ AA mice. Compared with control group, ELISA data showed that the levels of IL-6, IL-1ß and TNF-α in serum and skin tissues of CUMS induced AA mice were significantly increased, while XZD administration dramatically restrained the contents of the three pro-inflammatory factors. Western blot, immunohistochemistry, and qRT-PCR results further demonstrated that XZD administration notably down-regulated the protein and gene expression levels of osteopontin, IL-6, IL-1ß and TNF-α in comparation with CUMS group. CONCLUSION: XZD could dramatically ameliorate CUMS-induced AA damage in the skin of C3H/HeJ mice, possibly by suppressing the levels of IL-6, IL-1ß, TNF-α and osteopontin.

Biophysical and morphological changes in inner hair cells and their efferent innervation in the ageing mouse cochlea.

KEY POINTS: Age-related hearing loss is a progressive hearing loss involving environmental and genetic factors, leading to a decrease in hearing sensitivity, threshold and speech discrimination. We compared age-related changes in inner hair cells (IHCs) between four mouse strains with different levels of progressive hearing loss. The surface area of apical coil IHCs (9-12 kHz cochlear region) decreases by about 30-40% with age. The number of BK channels progressively decreases with age in the IHCs from most mouse strains, but the basolateral membrane current profile remains unchanged. The mechanoelectrical transducer current is smaller in mice harbouring the hypomorphic Cdh23 allele Cdh23ahl (C57BL/6J; C57BL/6NTac), but not in Cdh23-repaired mice (C57BL/6NTacCdh23+ ), indicating that it could contribute to the different progression of hearing loss among mouse strains. The degree of efferent rewiring onto aged IHCs, most likely coming from the lateral olivocochlea fibres, was correlated with hearing loss in the different mouse strains. ABSTRACT: Inner hair cells (IHCs) are the primary sensory receptors of the mammalian cochlea, transducing acoustic information into electrical signals that are relayed to the afferent neurons. Functional changes in IHCs are a potential cause of age-related hearing loss. Here, we have investigated the functional characteristics of IHCs from early-onset hearing loss mice harbouring the allele Cdh23ahl (C57BL/6J and C57BL/6NTac), from late-onset hearing loss mice (C3H/HeJ), and from mice corrected for the Cdh23ahl mutation (C57BL/6NTacCdh23+ ) with an intermediate hearing phenotype. There was no significant loss of IHCs in the 9-12 kHz cochlear region up to at least 15 months of age, but their surface area decreased progressively by 30-40% starting from ∼6 months of age. Although the size of the BK current decreased with age, IHCs retained a normal KCNQ4 current and resting membrane potential. These basolateral membrane changes were most severe for C57BL/6J and C57BL/6NTac, less so for C57BL/6NTacCdh23+ and minimal or absent in C3H/HeJ mice. We also found that lateral olivocochlear (LOC) efferent fibres re-form functional axon-somatic connections with aged IHCs, but this was seen only sporadically in C3H/HeJ mice. The efferent post-synaptic SK2 channels appear prior to the establishment of the efferent contacts, suggesting that IHCs may play a direct role in re-establishing the LOC-IHC synapses. Finally, we showed that the size of the mechanoelectrical transducer (MET) current from IHCs decreased significantly with age in mice harbouring the Cdh23ahl allele but not in C57BL/6NTacCdh23+ mice, indicating that the MET apparatus directly contributes to the progression of age-related hearing loss.

Chemokine expression profiles in liver and kidney of mice with different susceptibilities to leptospirosis.

Leptospirosis is a global disease that affects humans and animals, impacting public health and the economy. The symptoms caused by Leptospira infection can vary from mild to severe, affecting liver, lungs, and kidneys. The host-pathogen interaction in leptospirosis is still poorly understood, but there is evidence for the role of the host immune response in the pathogenesis. Chemokines are a family of structurally-related low-molecular-mass proteins (8-14 kDa) that signal the recruitment of leukocytes. In this study the profile of 22 chemokines were evaluated in liver and kidney of three mice strains with different phenotypes of susceptibility to leptospirosis. We extended our previously reported observations showing that expression of chemokines with homeostatic function, activation and chemotaxis of leukocytes are essential to modulate and to induce resistance to leptospirosis. Our findings support that an early induction of CXC chemokines in resistant BALB/c mice can be associated with the control of the infection. The correlation of chemokine expression between liver and kidney observed in BALB/c suggests that a balance of chemokine induction in the organs may contribute to resistance to leptospirosis.

Nonsurgical Induction of Alopecia Areata in C3H/HeJ Mice via Adoptive Transfer of Cultured Lymphoid Cells.

Surgical induction of alopecia areata (AA) via full-thickness grafting of spontaneous AA-affected C3H/HeJ mouse skin to naïve recipients has been a primary method of transferring the AA disease model phenotype. However, this method is associated with the need to perform an invasive procedure that could negatively impact animal wellbeing. Therefore, a rodent model that rapidly develops AA at a predictable rate and without the need to perform invasive surgical procedures on the mice is essential for studying the pathogenesis of AA. Here we describe a cell injection technique using cultured skin-draining lymph node cells (LNCs) injected intradermally into naïve recipients to induce rapid AA development. The cultured LNCs can reach ~ten fold expansion after 6 days with specific cytokine stimulation. The LNCs derived from a single AA affected mouse donor can induce AA development in more than 80 naïve mice within 2-18 weeks. For comparative control studies, mice receiving cultured LNCs from normal donors remain normally haired. The method enables the production of large numbers of AA mice for use in research and treatment development studies while avoiding the use of surgical procedures. We anticipate that the protocol can also be adapted for use in other mouse autoimmune disease models.

Plasmacytoid dendritic cells as a possible key player to initiate alopecia areata in the C3H/HeJ mouse.

BACKGROUND: Alopecia areata (AA) is a tissue-specific autoimmune disease, and interferon (IFN)-γ has been regarded as the key cytokine in the pathogenesis of AA. The clinical observation that AA can occur after viral infection or IFN-α administration implies that IFN-α-producing plasmacytoid dendritic cells (pDCs) may be involved in the AA pathogenesis. METHODS: We generated AA in C3H/HeJ mice by intradermal injection of T cells derived from lymph nodes of AA-bearing syngeneic mice and stimulated IL-2, IL-7, and IL-15. Distribution of IFN-γ producing pDCs were immunohistochemically analyzed. Realtime PCR were also demonstrated to detect the expression of IFN-γ mRNA. Hair follicles were cultured with IFN-α in order to calculate the hair elongation. Imiquimod was employed to induce catagen stage. PDCs were injected into C3H/HeJ mice to initiate AA. RESULTS: In this mouse, IFN-α-producing pDCs densely infiltrated around HFs in not only AA lesional but also vicinity of AA lesion. Importantly, intradermal injection of pDCs induced AA lesions. Finally, IFN-α inhibited hair elongation of murine vibrissae and upregulated MHC class I and CXCL10 levels in vitro. CONCLUSIONS: These findings suggest that IFN-α-producing pDCs initiate AA by inducing apoptosis and increasing Th1/Tc1 chemokine production such as CXCL10, that accumulates Th1/Tc1 cells and result in autoimmune reactions against hair follicles.

Chemokine expression profiles in liver and kidney of mice with different susceptibilities to leptospirosis

Leptospirosis is a global disease that affects humans and animals, impacting public health and the economy. The symptoms caused by Leptospira infection can vary from mild to severe, affecting liver, lungs, and kidneys. The host-pathogen interaction in leptospirosis is still poorly understood, but there is evidence for the role of the host immune response in the pathogenesis. Chemokines are a family of structurally-related low-molecular-mass proteins (8–14 kDa) that signal the recruitment of leukocytes. In this study the profile of 22 chemokines were evaluated in liver and kidney of three mice strains with different phenotypes of susceptibility to leptospirosis. We extended our previously reported observations showing that expression of chemokines with homeostatic function, activation and chemotaxis of leukocytes are essential to modulate and to induce resistance to leptospirosis. Our findings support that an early induction of CXC chemokines in resistant BALB/c mice can be associated with the control of the infection. The correlation of chemokine expression between liver and kidney observed in BALB/c suggests that a balance of chemokine induction in the organs may contribute to resistance to leptospirosis.

Abnormal Thyroid Hormone Status Differentially Affects Bone Mass Accrual and Bone Strength in C3H/HeJ Mice: A Mouse Model of Type I Deiodinase Deficiency.

C3H/HeJ (C3H) mice are deficient of type I deiodinase (D1), an enzyme that activates thyroid hormone (TH), converting thyroxine (T4) to triiodothyronine (T3). Nevertheless, C3H mice present normal serum T3 and a gross euthyroid phenotype. To investigate if a global D1 deficiency interferes in the TH effects on bone, we compared bone growth, bone mass accrual and bone strength of C3H and C57BL/6J (B6) mice under abnormal TH status. Four-week-old female mice of both strains were grouped as Euthyroid, Hypothyroid (pharmacologically-induced), 1xT4 and 10xT4 (hypothyroid animals receiving 1- or 10-fold the physiological dose of T4 /day/16 weeks). Hypothyroidism and TH excess similarly impaired body weight (BW) gain and body growth in both mice strains. In contrast, whereas hypothyroidism only slightly impaired bone mineral density (BMD) accrual in B6 mice, it severely impaired BMD accrual in C3H mice. No differences were observed in serum and bone concentrations of T3 between hypothyroid animals of both strains. Interestingly, treatment with 10xT4 was less deleterious to BMD accrual in C3H than in B6 mice and resulted in less elevated T3 serum levels in B6 than in C3H mice, which is probably explained by the lower D1 activity in C3H mice. In addition, hypothyroidism decreased bone strength only in C3H but not in B6 mice, while TH excess decreased this parameter in both strains. These findings indicate that D1 deficiency contributes to the TH excess-induced differences in bone mass accrual in C3H vs. B6 mice and suggest that deiodinase-unrelated genetic factors might account for the different skeleton responses to hypothyroidism between strains.

Phagocytosis of Leptospira by leukocytes from mice with different susceptibility to leptospirosis and possible role of chemokines.

BACKGROUND: Leptospirosis is a widespread zoonosis caused by pathogenic prokaryotic microbes of the genus Leptospira. Although there are several reports in the literature, host-pathogen interaction is still poorly understood. The role of chemokine expression is important on the chemotaxis, activation and regulation of immune cells. Recent studies have shown that their expression profiles play an important role on the severity of leptospirosis outcome. We evaluated the phagocytosis of Leptospira by spleens cells from C3H/HeJ, C3H/HePas and BALB/c mouse strains, respectively susceptible, intermediate and resistant to leptospirosis, and by RAW 264.7 macrophages. Besides, we evaluated the effects of CCL2 treatment on the phagocytosis. The cells were incubated with or without CCL2 chemokine, and infected with virulent L. interrogans sv Copenhageni. Cells and culture supernatants were collected for subsequent analysis. RESULTS: The number of leptospires was higher in BALB/c cells, CCL2 pre-treated or only infected groups, when compared to C3H/HeJ and C3H/HePas cells. Indeed, CCL2 activation did not interfere in the phagocytosis of Leptospira. Expression of chemokines CXCL5 and CCL8 levels were significantly inhibited in infected BALB/c cells when compared to the non-infected control. CONCLUSIONS: Higher ability to phagocytosis and early modulation of some chemokines correlated with the resistance to leptospirosis disease. Exposure to CCL2 did not interfere on phagocytosis of Leptospira in our experimental conditions, but acted in the modulation of chemokines expression during Leptospira infection.

The effect of PPARγ inhibition on bone marrow adipose tissue and bone in C3H/HeJ mice.

Bone marrow adipose tissue (BMAT) increases after menopause, and increased BMAT is associated with osteoporosis and prevalent vertebral fractures. Peroxisome proliferator-activated receptor-γ (PPARγ) activation promotes adipogenesis and inhibits osteoblastogenesis; therefore, PPARγ is a potential contributor to the postmenopausal increase in BMAT and decrease in bone mass. The aim of this study is to determine if PPARγ inhibition can prevent ovariectomy-induced BMAT increase and bone loss in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice ( n = 40) were allocated to four intervention groups: sham surgery (Sham) or ovariectomy (OVX; isoflurane anesthesia) with either vehicle (Veh) or PPARγ antagonist administration (GW9662; 1 mg·kg-1·day-1, daily intraperitoneal injections) for 3 wk. We measured BMAT volume, adipocyte size, adipocyte number. and bone structural parameters in the proximal metaphysis of the tibia using polyoxometalate-based contrast enhanced-nanocomputed topogaphy. Bone turnover was measured in the contralateral tibia using histomorphometry. The effects of surgery and treatment were analyzed by two-way ANOVA. OVX increased the BMAT volume fraction (Sham + Veh: 2.9 ± 2.7% vs. OVX + Veh: 8.1 ± 5.0%: P < 0.001), average adipocyte diameter (Sham + Veh: 19.3 ± 2.6 µm vs. OVX + Veh: 23.1 ± 3.4 µm: P = 0.001), and adipocyte number (Sham + Veh: 584 ± 337cells/µm3 vs. OVX + Veh: 824 ± 113cells/µm3: P = 0.03), while OVX decreased bone volume fraction (Sham + Veh: 15.5 ± 2.8% vs. OVX + Veh: 7.7 ± 1.9%; P < 0.001). GW9662 had no effect on BMAT, bone structural parameters, or bone turnover. In conclusion, ovariectomy increased BMAT and decreased bone volume in C3H/HeJ mice. The PPARγ antagonist GW9662 had no effect on BMAT or bone volume in C3H/HeJ mice, suggesting that BMAT accumulation is regulated independently of PPARγ in C3H/HeJ mice.

Phagocytosis of Leptospira by leukocytes from mice with different susceptibility to leptospirosis and possible role of chemokines

Background: Leptospirosis is a widespread zoonosis caused by pathogenic prokaryotic microbes of the genus Leptospira. Although there are several reports in the literature, host-pathogen interaction is still poorly understood. The role of chemokine expression is important on the chemotaxis, activation and regulation of immune cells. Recent studies have shown that their expression profiles play an important role on the severity of leptospirosis outcome. We evaluated the phagocytosis of Leptospira by spleens cells from C3H/HeJ, C3H/HePas and BALB/c mouse strains, respectively susceptible, intermediate and resistant to leptospirosis, and by RAW 264.7 macrophages. Besides, we evaluated the effects of CCL2 treatment on the phagocytosis. The cells were incubated with or without CCL2 chemokine, and infected with virulent L. interrogans sv Copenhageni. Cells and culture supernatants were collected for subsequent analysis. Results: The number of leptospires was higher in BALB/c cells, CCL2 pre-treated or only infected groups, when compared to C3H/HeJ and C3H/HePas cells. Indeed, CCL2 activation did not interfere in the phagocytosis of Leptospira. Expression of chemokines CXCL5 and CCL8 levels were significantly inhibited in infected BALB/c cells when compared to the non-infected control. Conclusions: Higher ability to phagocytosis and early modulation of some chemokines correlated with the resistance to leptospirosis disease. Exposure to CCL2 did not interfere on phagocytosis of Leptospira in our experimental conditions, but acted in the modulation of chemokines expression during Leptospira infection.

Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies using contemporary criteria.

Skin tumors have been observed in C3H/HeJ mice following treatment with high and strongly irritating concentrations of 2-ethylhexyl acrylate (2-EHA). Dermal carcinogenicity studies performed with 2-EHA are reviewed, contrasting the results in two mouse strains (C3H/HeJ and NMRI) under different dosing regimens. Application of contemporary evaluation criteria to the existing dermal carcinogenicity dataset demonstrates that 2-EHA induces skin tumors only at concentrations exceeding an maximum tolerated dose (MTD) and in the immune-dysregulated C3H/HeJ mouse model. Overall, the available chronic toxicity and genotoxicity data on 2-EHA support a non-genotoxic chemical irritant mechanism, whereby chronic irritation leads to inflammation, tissue injury, and wound repair, the latter of which is disrupted in C3H/HeJ mice and leads to tumor formation. Tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal carcinogenesis in an immune competent (NMRI) mouse model, and, even in the immune compromised C3H/HeJ model, did not induce skin tumors at doses which did not exceed the MTD.

Resistance of mice to Leptospira infection and correlation with chemokine response.

Leptospirosis is globally widespread neglected disease, affecting most mammalian species. Clinical signs can be confused with other diseases which make the diagnosis and treatment difficult. Chemokines and cytokines are known for their role in the inflammatory and immune response to infections. The profile determination of chemokines' expressions in the course of infection may elucidate the defense mechanisms of the host and support the search for effective treatment strategies. We investigated the mechanisms of innate immunity through the comparison of chemokines induced during infection with L. interrogans in mice with different levels of susceptibility. We used lung and spleen tissues samples of mice from C3H/HeJ, C3H/HePas and Balb/c, respectively sensitive, intermediate susceptibility and resistant to the pathogen. The inoculation of L. interrogans in C3H/HeJ mice led a comparatively smaller change in chemokines expression in both spleen and lung tissues. In samples from spleens and lungs of C3H/HePas and Balb/c the higher increases occurred on CXCL9, CXCL16, CXCL5, CCL8 and CCL5 in Balb/c. Given the same genetic background, the differences in the responses of C3H/HePas compared to C3H/HeJ mice strongly suggest the role of chemokines for the survival of parental strain. Therefore, the greatest increase in CXC chemokines appears to be efficient to induce migration of cells to the secondary lymphoid organs and affected tissues, which is important to control infection. Overall, CXC chemokines are important for the activation and attraction of T cell and may influence the course and control of the infection in resistant Balb/c mice.

Eyedrop Inoculation Causes Sublethal Leptospirosis in Mice.

Leptospirosis is potentially a fatal zoonosis acquired by contact of skin and mucosal surfaces with soil and water contaminated with infected urine. We analyzed the outcome of infection of C3H/HeJ mice with Leptospira interrogans serovar Copenhageni using an enzootic mode of transmission, the conjunctival route. Infection led to weight loss and L. interrogans dissemination from blood to urine, and spirochetes were detected in blood and urine simultaneously. The infectious dose that led to consistent dissemination to kidney after conjunctival infection was ∼108 leptospires. Interestingly, a lower number of spirochetes appeared to colonize the kidney, given that we quantified ∼105 and ∼10 leptospires per µl of urine and per µg of kidney, respectively. Leptospira-specific IgM and IgG were detected at 15 days postinfection, and isotyping of the Ig subclass showed that the total IgG response switched from an IgG1 response to an IgG3 response after infection with L. interrogans Histological periodic acid-Schiff D staining of infected kidney showed interstitial nephritis, mononuclear cell infiltrates, and reduced size of glomeruli. Quantification of proinflammatory immunomediators in kidney showed that keratinocyte-derived chemokine, macrophage inflammatory protein 2, RANTES, tumor necrosis factor alpha, gamma interferon, and interleukin-10 were upregulated in infected mice. We show that the kinetics of disease progression after infection via the ocular conjunctiva is delayed compared with infection via the standard intraperitoneal route. Differences may be related to the number of L. interrogans spirochetes that succeed in overcoming the natural defenses of the ocular conjunctiva and transit through tissue.

Curdlan activates dendritic cells through dectin-1 and toll-like receptor 4 signaling.

Curdlan, a ß-1,3-glucan isolated from Alcaligenes faecalis, is an agonist of dectin-1 in various immune cells, including dendritic cells (DCs). However, whether curdlan also activates DCs through other receptors remains unknown. In this study, we found that curdlan activates DCs through dectin-1 and toll-like receptor 4 (TLR4). Curdlan increased the expression levels of surface molecules (CD40, CD80, CD86, and MHC-I/II), the production of cytokines (IL-12, IL-1ß, TNF-α, and IFN-ß), migration toward MIP-3ß, and allogeneic T cell stimulation activity of DCs. Curdlan increased the phosphorylation of Syk, Raf-1, Akt, MAPKs, IKK, and NF-κB p65 in DCs. However, curdlan only slightly activated DCs transfected with small interfering RNAs against dectin-1 or TLR4 and C3H/HeJ DCs, which have non-functional TLR4, in comparison with control DCs. Curdlan increased antitumor activity of DCs in a syngeneic tumor model. In summary, our data show that curdlan activates DCs through dectin-1 and TLR4 signaling and the combination of curdlan and DCs efficiently inhibit tumor growth in mice.

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration.

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.

Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4.

Our laboratory develops protocols to prevent or reverse ongoing anti-hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9(-/Y) mice develop high titre anti-hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9(-/Y) mice have mild anti-hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX-specific B- and T-cell responses in BALB/c and C3H/HeJ F9(-/Y) mice trigger the difference in anti-hFIX immune responses. BALB/c and C3H/HeJ F9(-/Y) mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti-hFIX ELISA, Bethesda assay for inhibitors and B-cell ELISpot on bone marrow and spleen cells. T-cell studies measured the TH 1 (IFN-γ) and TH 2 (IL-4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9(-/Y) mice with restored toll-like receptor 4 (TLR4) function. BALB/c F9(-/Y) mice have a TH 2 skewed response and a reduction in anti-hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9(-/Y) mice have a mixed TH 1 and TH 2 response (mainly TH 2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti-hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.