Improved corneal clarity following lamellar keratectomy for corneal lipidosis in a canine with ocular manifestations of hypothyroidism.

OBJECTIVE: To report the corneal clarity outcome following lamellar keratectomy of arcus lipoides corneae secondary to canine hypothyroidism and report a unique retinal manifestation of systemic disease. ANIMAL STUDIED: Four-year-old spayed female Sheepdog-Poodle canine. PROCEDURE: Lamellar keratectomy OD. RESULTS: Bilateral severe arcus lipoides corneae was noted in the initial presentation. Bilateral, symmetric, and multifocal bullous retinal detachments were observed at subsequent visits. Biochemical testing revealed hyperlipidemia presumed to be associated with primary acquired thyroiditis. Corneal clarity and visual behaviors were significantly improved following unilateral lamellar keratectomy with no evidence of recurrence within the year following surgery. Bilateral retinal detachments and hyperlipidemia resolved months after initiation of thyroxine supplementation. Corneal lipidosis in the untreated eye remained static. CONCLUSIONS: Lamellar keratectomy is a viable surgical option for the treatment of arcus lipoides corneae. Hypothyroidism should be considered a differential diagnosis for spontaneous, bilateral, multifocal, and serous retinal detachments.

Tlr5 deficiency exacerbates lupus-like disease in the MRL/lpr mouse model.

Introduction: Leaky gut has been linked to autoimmune disorders including lupus. We previously reported upregulation of anti-flagellin antibodies in the blood of lupus patients and lupus-prone mice, which led to our hypothesis that a leaky gut drives lupus through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5). Methods: We created MRL/lpr mice with global Tlr5 deletion through CRISPR/Cas9 and investigated lupus-like disease in these mice. Result: Contrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus with Tlr5 deficiency in female MRL/lpr mice. Remarkably higher levels of proteinuria were observed in Tlr5 -/- MRL/lpr mice suggesting aggravated glomerulonephritis. Histopathological analysis confirmed this result, and Tlr5 deletion significantly increased the deposition of IgG and complement C3 in the glomeruli. In addition, Tlr5 deficiency significantly increased renal infiltration of Th17 and activated cDC1 cells. Splenomegaly and lymphadenopathy were also aggravated in Tlr5-/- MRL/lpr mice suggesting impact on lymphoproliferation. In the spleen, significant decreased frequencies of regulatory lymphocytes and increased germinal centers were observed with Tlr5 deletion. Notably, Tlr5 deficiency did not change host metabolism or the existing leaky gut; however, it significantly reshaped the fecal microbiota. Conclusion: Global deletion of Tlr5 exacerbates lupus-like disease in MRL/lpr mice. Future studies will elucidate the underlying mechanisms by which Tlr5 deficiency modulates host-microbiota interactions to exacerbate lupus.

TCDD and CH223191 Alter T Cell Balance but Fail to Induce Anti-Inflammatory Response in Adult Lupus Mice.

Aryl hydrocarbon receptor (AhR) responds to endogenous and exogenous ligands as a cytosolic receptor, transcription factor, and E3 ubiquitin ligase. Several studies support an anti-inflammatory effect of AhR activation. However, exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early stages of development results in an autoimmune phenotype and exacerbates lupus. The effects of TCDD on lupus in adults with pre-existing autoimmunity have not been described. We present novel evidence that AhR stimulation by TCDD alters T cell responses but fails to impact lupus-like disease using an adult mouse model. Interestingly, AhR antagonist CH223191 also changed T cell balance in our model. We next developed a conceptual framework for identifying cellular and molecular factors that contribute to physiological outcomes in lupus and created models that describe cytokine dynamics that were fed into a system of differential equations to predict the kinetics of T follicular helper (Tfh) and regulatory T (Treg) cell populations. The model predicted that Tfh cells expanded to larger values following TCDD exposure compared with vehicle and CH223191. Following the initial elevation, both Tfh and Treg cell populations continuously decayed over time. A function based on the ratio of predicted Treg/Tfh cells showed that Treg cells exceed Tfh cells in all groups, with TCDD and CH223191 showing lower Treg/Tfh cell ratios than the vehicle and that the ratio is relatively constant over time. We conclude that AhR ligands did not induce an anti-inflammatory response to attenuate autoimmunity in adult lupus mice. This study challenges the dogma that TCDD supports an immunosuppressive phenotype.

Xanthogranulomatous keratitis in a mixed-breed dog.

OBJECTIVE: To evaluate the clinical and histopathological features of a case of xanthogranulomatous keratitis in a mixed-breed dog. ANIMAL: Mixed-breed dog. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: An 11-year-old spayed female mixed-breed dog was presented for mild blepharospasm, corneal cloudiness, and increasing conjunctival hyperemia OD. Ophthalmic examination revealed multifocal pink and cream-colored consolidated corneal infiltrative lesions and generalized neovascularization with suspected diagnosis of stromal abscessation. There was no improvement after 1 month of medical management, so a keratectomy was performed, and corneal tissue was sent for histopathological evaluation. TREATMENT AND OUTCOME: The nonulcerative keratitis was refractive to medical management including topical and systemic antibiotics, topical antifungal, and systemic anti-inflammatory, so keratectomy was performed. Histopathologic diagnosis of xanthogranulomatous keratitis was made 1 week postoperatively. The patient was prescribed 0.05% difluprednate ophthalmic emulsion and 0.2% tacrolimus ophthalmic ointment (initially q 8 h, OD). The difluprednate was tapered and discontinued after 2 months, but the tacrolimus was continued (q 12 h, OD). No lesion recurrence had been documented 1 year postoperatively. CLINICAL RELEVANCE: There has been little published on canine xanthogranulomas, especially in veterinary ophthalmology. Ocular xanthogranulomas have been reportedly found intraocularly and at the ocular surface. Histologically, they are characterized by well-delineated nodules that contain histiocytes and abundant lipid-laden macrophages. The treatment in this clinical case was surgical excision followed with topical immunosuppression/anti-inflammatory therapy with no recurrence 1 year postoperatively. Xanthogranulomatous keratitis should be an added differential diagnosis when nonulcerative keratitis is found on examination, specifically with consolidated, corneal infiltrate and minimal pain.

Egr2 Deletion in Autoimmune-Prone C57BL6/lpr Mice Suppresses the Expression of Methylation-Sensitive Dlk1-Dio3 Cluster MicroRNAs.

We previously demonstrated that the upregulation of microRNAs (miRNAs) at the genomic imprinted Dlk1-Dio3 locus in murine lupus is correlated with global DNA hypomethylation. We now report that the Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice is hypomethylated, linking it to increased Dlk1-Dio3 miRNA expression. We evaluated the gene expression of methylating enzymes, DNA methyltransferases (DNMTs), and demethylating ten-eleven translocation proteins (TETs) to elucidate the molecular basis of DNA hypomethylation in lupus CD4+ T cells. There was a significantly elevated expression of Dnmt1 and Dnmt3b, as well as Tet1 and Tet2, in CD4+ T cells of three different lupus-prone mouse strains compared to controls. These findings suggest that the hypomethylation of murine lupus CD4+ T cells is likely attributed to a TET-mediated active demethylation pathway. Moreover, we found that deletion of early growth response 2 (Egr2), a transcription factor gene in B6/lpr mice markedly reduced maternally expressed miRNA genes but not paternally expressed protein-coding genes at the Dlk1-Dio3 locus in CD4+ T cells. EGR2 has been shown to induce DNA demethylation by recruiting TETs. Surprisingly, we found that deleting Egr2 in B6/lpr mice induced more hypomethylated differentially methylated regions at either the whole-genome level or the Dlk1-Dio3 locus in CD4+ T cells. Although the role of methylation in EGR2-mediated regulation of Dlk1-Dio3 miRNAs is not readily apparent, these are the first data to show that in lupus, Egr2 regulates Dlk1-Dio3 miRNAs, which target major signaling pathways in autoimmunity. These data provide a new perspective on the role of upregulated EGR2 in lupus pathogenesis.

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by environmental factors and loss of key proteins, including the endonuclease Dnase1L3. Dnase1L3 absence causes pediatric-onset lupus in humans, while reduced activity occurs in adult-onset SLE. The amount of Dnase1L3 that prevents lupus remains unknown. To genetically reduce Dnase1L3 levels, we developed a mouse model lacking Dnase1L3 in macrophages (conditional knockout [cKO]). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Homogeneous and peripheral antinuclear antibodies were detected in the sera by immunofluorescence, consistent with anti-double-stranded DNA (anti-dsDNA) antibodies. Total immunoglobulin M, total immunoglobulin G, and anti-dsDNA antibody levels increased in cKO mice with age. The cKO mice developed anti-Dnase1L3 antibodies. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated early in life. The cKO mice had minimal kidney pathology. Therefore, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes, and macrophage-derived DnaselL3 helps limit lupus.

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deleting Dnase1L3 from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3 -/- mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

CX3CR1 modulates SLE-associated glomerulonephritis and cardiovascular disease in MRL/lpr mice.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) often develop multi-organ damages including heart and kidney complications. We sought to better define the underlying mechanisms with a focus on the chemokine receptor CX3CR1. METHODS: We generated Cx3cr1-deficient MRL/lpr lupus-prone mice through backcrossing. We then employed heterozygous intercross to generate MRL/lpr littermates that were either sufficient or deficient of CX3CR1. The mice were also treated with either Lactobacillus spp. or a high-fat diet (HFD) followed by assessments of the kidney and heart, respectively. RESULTS: Cx3cr1-/- MRL/lpr mice exhibited a distinct phenotype of exacerbated glomerulonephritis compared to Cx3cr1+/+ littermates, which was associated with a decrease of spleen tolerogenic marginal zone macrophages and an increase of double-negative T cells. Interestingly, upon correction of the gut microbiota with Lactobacillus administration, the phenotype of exacerbated glomerulonephritis was reversed, suggesting that CX3CR1 controls glomerulonephritis in MRL/lpr mice through a gut microbiota-dependent mechanism. Upon treatment with HFD, Cx3cr1-/- MRL/lpr mice developed significantly more atherosclerotic plaques that were promoted by Ly6C+ monocytes. Activated monocytes expressed ICOS-L that interacted with ICOS-expressing follicular T-helper cells, which in turn facilitated a germinal center reaction to produce more autoantibodies. Through a positive feedback mechanism, the increased circulatory autoantibodies further promoted the activation of Ly6C+ monocytes and their display of ICOS-L. CONCLUSIONS: We uncovered novel, Cx3cr1 deficiency-mediated pathogenic mechanisms contributing to SLE-associated glomerulonephritis and cardiovascular disease.

Hypovitaminosis A Drives the Progression of Tubulointerstitial Lupus Nephritis through Potentiating Predisease Cellular Autoreactivity.

Vitamin A (VA) deficiency (VAD) is observed in both humans and mice with lupus nephritis. However, whether VAD is a driving factor for accelerated progression of lupus nephritis is unclear. In this study, we investigated the effect of VAD on the progression of lupus nephritis in a lupus-prone mouse model, MRL/lpr. We initiated VAD either during gestation or after weaning to reveal a potential time-dependent effect. We found exacerbated lupus nephritis at ∼15 wk of age with both types of VAD that provoked tubulointerstitial nephritis leading to renal failure. This was concomitant with significantly higher mortality in all VAD mice. Importantly, restoration of VA levels after weaning reversed VAD-induced mortality. These results suggest VAD-driven acceleration of tubulointerstitial lupus nephritis. Mechanistically, at the earlier time point of 7 wk of age and before the onset of clinical lupus nephritis, continued VAD (from gestation until postweaning) enhanced plasma cell activation and augmented their autoantibody production, while also increasing the expansion of T lymphocytes that could promote plasma cell autoreactivity. Moreover, continued VAD increased the renal infiltration of plasmacytoid dendritic cells. VAD initiated after weaning, in contrast, showed modest effects on autoantibodies and renal plasmacytoid dendritic cells that were not statistically significant. Remarkably, analysis of gene expression in human kidney revealed that the retinoic acid pathway was decreased in the tubulointerstitial region of lupus nephritis, supporting our findings in MRL/lpr mice. Future studies will elucidate the underlying mechanisms of how VAD modulates cellular functions to exacerbate tubulointerstitial lupus nephritis.

A double-edged sword: interactions of CX3CL1/CX3CR1 and gut microbiota in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic chronic disease initiated by an abnormal immune response to self and can affect multiple organs. SLE is characterized by the production of autoantibodies and the deposition of immune complexes. In regard to the clinical observations assessed by rheumatologists, several chemokines and cytokines also contribute to disease progression. One such chemokine and adhesion molecule is CX3CL1 (otherwise known as fractalkine). CX3CL1 is involved in cell trafficking and inflammation through recognition by its receptor, CX3CR1. The CX3CL1 protein consists of a chemokine domain and a mucin-like stalk that allows it to function both as a chemoattractant and as an adhesion molecule. In inflammation and specifically lupus, the literature displays contradictory evidence for the functions of CX3CL1/CX3CR1 interactions. In addition, the gut microbiota has been shown to play an important role in the pathogenesis of SLE. This review highlights current studies that illustrate the interactions of the gut microbiota and CX3CR1 in SLE.

Lactobacillus spp. act in synergy to attenuate splenomegaly and lymphadenopathy in lupus-prone MRL/lpr mice.

Commensal bacteria and the immune system have a close and strong relationship that maintains a balance to control inflammation. Alterations of the microbiota, known as dysbiosis, can direct reactivity to self-antigens not only in the intestinal mucosa but also at the systemic level. Our laboratory previously reported gut dysbiosis, particularly lower abundance of bacteria in the family Lactobacillaceae, in lupus-prone MRL/lpr mice, a model of systemic autoimmunity. Restoring the microbiota with a mix of 5 different Lactobacillus species (spp.), L. reuteri, L. oris, L. johnsonii, L. gasseri and L. rhamnosus, attenuated lupus-liked clinical signs, including splenomegaly and lymphadenopathy. However, our understanding of the mechanism was limited. In this study, we first investigated the effects of individual species. Surprisingly, none of the species individually recapitulated the benefits of the mix. Instead, Lactobacillus spp. acted synergistically to attenuate splenomegaly and renal lymphadenopathy through secreted factors and a CX3CR1-dependent mechanism. Interestingly, oral administration of MRS broth exerted the same benefits likely through increasing the relative abundance of endogenous Lactobacillus spp. Mechanistically, we found increased percentages of FOXP3-negative type 1 regulatory T cells with administration of the mix in both spleen and mesenteric lymph nodes. In addition, oral gavage of Lactobacillus spp. decreased the percentage of central memory T cells while increasing that of effector memory T cells in the lymphoid organs. Furthermore, a decreased percentage of double negative T cells was observed in the spleen with the mix. These results suggest that Lactobacillus spp. might act on T cells to attenuate splenomegaly and lymphadenopathy. Together, this study advances our understanding of how Lactobacillus spp. attenuate lupus in MRL/lpr mice. The synergistic action of these bacteria suggests that multiple probiotic bacteria in combination may dampen systemic autoimmunity and benefit lupus patients.

Deletion of microRNA-183-96-182 Cluster in Lymphocytes Suppresses Anti-DsDNA Autoantibody Production and IgG Deposition in the Kidneys in C57BL/6-Faslpr/lpr Mice.

Dysregulated miRNAs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study reported a substantial increase in three miRNAs located at the miR-183-96-182 cluster (miR-183C) in several autoimmune lupus-prone mice, including MRL/lpr and C57BL/6-lpr (B6/lpr). This study reports that in vitro inhibition of miR-182 alone or miR-183C by specific antagomirs in activated splenocytes from autoimmune-prone MRL/lpr and control MRL mice significantly reduced lupus-related inflammatory cytokines, interferon-gamma (IFNγ), and IL-6 production. To further characterize the role of miR-182 and miR-183C cluster in vivo in lupus-like disease and lymphocyte phenotypes, we used hCD2-iCre to generate B6/lpr mice with conditional deletion of miR-182 or miR-183C in CD2+ lymphocytes (miR-182-/-B6/lpr and miR-183C-/-B6/lpr). The miR-182-/-B6/lpr and miR-183C-/-B6/lpr mice had significantly reduced deposition of IgG immunocomplexes in the kidney when compared to their respective littermate controls, although there appeared to be no remarkable changes in renal pathology. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C-/-B6/lpr mice after reaching 24 weeks-of age compared to age-matched miR-183Cfl/flB6/lpr controls. In vitro activated splenocytes from miR-182-/-B6/lpr mice and miR-183C-/-B6/lpr mice showed reduced ability to produce lupus-associated IFNγ. Forkhead box O1(Foxo1), a previously validated miR-183C miRNAs target, was increased in the splenic CD4+ cells of miR-182-/-B6/lpr and miR-183C-/-B6/lpr mice. Furthermore, in vitro inhibition of Foxo1 with siRNA in splenocytes from miR-182-/-B6/lpr and miR-183C-/-B6/lpr mice significantly increased IFNγ expression following anti-CD3/CD28 stimulation, suggesting that miR-182 and miR-183C miRNAs regulate the inflammatory IFNγ in splenocytes via targeting Foxo1. The deletion of either miR-182 alone or the whole miR-183C cluster, however, had no marked effect on the composition of T and B cell subsets in the spleens of B6/lpr mice. There were similar percentages of CD4+, CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells in the miR-183C-/-B6/lpr and miR-182-/-B6/lpr mice and their respective littermate controls, miR-183Cfl/flB6/lpr and miR-182fl/flB6/lpr mice. Together, our data demonstrate a role of miR-183C in the regulation of anti-dsDNA autoantibody production in vivo in B6/lpr mice and the induction of IFNγ in in vitro activated splenocytes from B6/lpr mice.

EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice.

Previous studies have reported that deletion of the transcription factor, early growth response protein 2 (EGR2), in normal C57BL/6 (B6) resulted in the development of lupus-like autoimmune disease. However, increased EGR2 expression has been noted in human and murine lupus, which challenges the notion of the autoimmune suppressive role of EGR2 in B6 mice. In this study, we derived both conditional EGR2-/-B6/lpr and EGR2-/-B6 mice to elucidate the immune and autoimmune regulatory roles of EGR2 in autoinflammation (B6/lpr) versus physiologically normal (B6) conditions. We found that conditional EGR2 deletion increased spleen weight, enhanced T cell activation and IFNγ production, and promoted germinal center B cells and LAG3+ regulatory T cells development in both B6/lpr and B6 mice. Nevertheless, EGR2 deletion also showed strikingly differential effects in these two strains on T lymphocyte subsets profile, Foxp3+ Tregs and plasma cell differentiation, anti-dsDNA autoantibodies and immunoglobulins production, and on the induction of IL-17 in in vitro activated splenocytes. Specifically, EGR2 deletion in B6/lpr mice significantly decreased serum levels of anti-dsDNA autoantibodies, total IgG, IgM, IgG1, and IgG2a with reduced plasma cells differentiation. Furthermore, EGR2 deletion in B6/lpr mice had no obvious effect on IgG immunocomplex deposition, medium caliber vessel, and glomeruli inflammation but increased complement C3 immunocomplex deposition and large caliber vessel inflammation in the kidneys. Importantly, we demonstrated that EGR2 deletion in B6/lpr mice significantly reduced pathogenic CD4-CD8-CD3+B220+ double negative T cells, which correlated with the reduced anti-dsDNA autoantibodies in serum and decreased IL-17 production in splenocytes of EGR2-/-B6/lpr mice. Together, our data strongly suggest that the role of EGR2 is complex. The immunoregulatory role of EGR2 varies at normal or autoinflammation conditions and should not be generalized in differential experimental settings.

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice using a Simple, Cost-Effective DNA Isolation Method.

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

Altered Germinal-Center Metabolism in B Cells in Autoimmunity.

B lymphocytes play an important role in the pathophysiology of many autoimmune disorders by producing autoantibodies, secreting cytokines, and presenting antigens. B cells undergo extreme physiological changes as they develop and differentiate. Aberrant function in tolerogenic checkpoints and the metabolic state of B cells might be the contributing factors to the dysfunctionality of autoimmune B cells. Understanding B-cell metabolism in autoimmunity is important as it can give rise to new treatments. Recent investigations have revealed that alterations in metabolism occur in the activation of B cells. Several reports have suggested that germinal center (GC) B cells of individuals with systemic lupus erythematosus (SLE) have altered metabolic function. GCs are unique microenvironments in which the delicate and complex process of B-cell affinity maturation occurs through somatic hypermutation (SHM) and class switching recombination (CSR) and where Bcl6 tightly regulates B-cell differentiation into memory B-cells or plasma cells. GC B cells rely heavily on glucose, fatty acids, and oxidative phosphorylation (OXPHOS) for their energy requirements. However, the complicated association between GC B cells and their metabolism is still not clearly understood. Here, we review several studies of B-cell metabolism, highlighting the significant transformations that occur in GC progression, and suggest possible approaches that may be investigated to more precisely target aberrant B-cell metabolism in SLE.

Phenotypic Drift in Lupus-Prone MRL/lpr Mice: Potential Roles of MicroRNAs and Gut Microbiota.

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors. We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation.

Changing the Wound: Covalent Immobilization of the Epidermal Growth Factor.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.

Regulation of neonatal IgA production by the maternal microbiota.

Infants are prone to enteric infections due to an underdeveloped immune system. The maternal microbiota, through shaping the neonatal microbiota, helps establish a strong immune system in infants. We and others have observed the phenomenon of enhanced early neonatal immunoglobulin A (IgA) production in preweaning immunocompetent mice nursed by immunodeficient dams. Here, we show that this enhancement of IgA in neonates results from maternally derived microbiota. In addition, we have found that the neonatal IgA production can be induced by Lactobacillus reuteri, which is enriched in the milk of immunodeficient dams. Moreover, we show that while the production of neonatal IgA is dependent on neonatal T cells, the immunodeficient maternal microbiota-mediated enhancement of neonatal IgA has a T cell-independent component. Indeed, this enhancement may be dependent on type 3 innate lymphoid cells in the neonatal small intestinal lamina propria. Interestingly, maternal microbiota-induced neonatal IgA does not cross-react with common enteric pathogens. Future investigations will determine the functional consequences of having this extra IgA.

Choroidal neuroendocrine neoplasia in a dog.

OBJECTIVE: To report onset and progression of clinical signs of a neuroendocrine neoplasm (NEN) presumed metastatic to the choroid in a dog. ANIMALS STUDIED: A 7.5-year-old female spayed German shepherd dog mix referred for advanced imaging and evaluation of a subretinal mass in the right eye. PROCEDURES: Procedures performed included general physical and ophthalmic examinations; ocular, orbital, and abdominal ultrasonography; thoracic radiographs; cranial magnetic resonance imaging; serologic testing for infectious agents; analysis of hematologic as well as serum and urine biochemical parameters; echocardiography; electrocardiography; cytologic assessment of lymph nodes; and histopathology and immunohistochemistry of the enucleated globe. RESULTS: Examination and imaging identified a pigmented mass within and expanding the superior choroid. Following enucleation, a choroidal NEN with tumor emboli in scleral blood vessels was diagnosed by histopathologic assessment and confirmed by immunohistochemical labelling. Despite extensive and repeated diagnostic testing over many months, a putative primary site was not identified until 19 months after the initial ocular signs were noted. At that time, a heart-base mass and congestive heart failure were highly suggestive of a chemodectoma. CONCLUSION: This comprehensive report of a NEN presumed metastatic to the choroid in a dog suggests that ocular disease can be a very early and solitary sign of NEN in the dog.