Abrogated AID Function Prolongs Survival and Diminishes Renal Pathology in the BXSB Mouse Model of Systemic Lupus Erythematosus.

Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex-mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73+ B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.

Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4+ T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4+ T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4+ T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4+ T cells were introduced into Rag1-/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4+ T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4+ T cells in a nonredundant manner with myeloid/stromal cells.

Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn-albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.

2-deoxy D-glucose treatment does not elicit a hair growth response in alopecia areata.

2-deoxy D-glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T-cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell-mediated autoimmune mouse disease, alopecia areata.

Skin fragility in the wild-derived, inbred mouse strain Mus pahari/EiJ.

Mus pahari is a wild-derived, inbred mouse strain. M. pahari colony managers observed fragility of this strain's skin resulting in separation of tail skin from the mouse if handled incorrectly. Tail skin tension testing of M. pahari resulted in significantly lowered force threshold for caudal skin rupture and loss in comparison to closely related inbred mouse species and subspecies and even more than a model for junctional epidermolysis bullosa. Histologically, the tail skin separated at the subdermal level with the dermis firmly attached to the epidermis, excluding the epidermolysis bullosa complex of diseases. The dermal collagen bundles were abnormally thickened and branched. Elastin fiber deposition was focally altered in the dermis adjacent to the hair follicle. Collagens present in the skin could not be differentiated between the species in protein gels following digestion with pepsin. Together these data suggest that M. pahari have altered extracellular matrix development resulting in separation of the skin below the level of the dermis with moderate force similar to the African spiny mouse (Acomys spp.).

Molecular identification of collagen 17a1 as a major genetic modifier of laminin gamma 2 mutation-induced junctional epidermolysis bullosa in mice.

Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2(jeb) mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2(jeb) mice. This modifier is defined by variations in 1-3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2(jeb) mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.

Minor genomic differences between related B6 and B10 mice affect severity of schistosome infection by governing the mode of dendritic cell activation.

Infection with the helminth Schistosoma mansoni results in hepatointestinal granulomatous inflammation mediated by CD4 T cells directed against parasite eggs. The severity of disease varies greatly in humans and mice; however, the genetic basis of such a heterogenous immune response remains poorly understood. Here we show that, despite their close genetic relationship, C57BL/10SnJ (B10) mice developed significantly more pronounced immunopathology and higher T helper 17 cell responses than C57BL/6J (B6) mice. Similarly, live egg-stimulated B10-derived dendritic cells (DCs) produced significantly more IL-1ß and IL-23, resulting in higher IL-17 production by CD4 T cells. Gene expression analysis disclosed a heightened proinflammatory cytokine profile together with a strikingly lower expression of Ym1 in B10 versus B6 mice, consistent with failure of B10 DCs to attain alternative activation. To genetically dissect the differential response, we developed and analyzed congenic mouse strains that capture major regions of allelic variation, and found that the level of inflammation was controlled by a relatively small number of genes in a locus mapping to chromosome 4 117-143 MB. Our study has thus identified novel genomic regions that regulate the severity of the schistosome infection by way of controlling the mode of DC activation and consequent CD4 T-cell subset development.

IL-21-driven neoplasms in SJL mice mimic some key features of human angioimmunoblastic T-cell lymphoma.

SJL/J mice exhibit a high incidence of mature B-cell lymphomas that require CD4(+) T cells for their development. We found that their spleens and lymph nodes contained increased numbers of germinal centers and T follicular helper (TFH) cells. Microarray analyses revealed high levels of transcripts encoding IL-21 associated with high levels of serum IL-21. We developed IL-21 receptor (IL21R)-deficient Swiss Jim Lambart (SJL) mice to determine the role of IL-21 in disease. These mice had reduced numbers of TFH cells, lower serum levels of IL-21, and few germinal center B cells, and they did not develop B-cell tumors, suggesting IL-21-dependent B-cell lymphomagenesis. We also noted a series of features common to SJL disease and human angioimmunoblastic T-cell lymphoma (AITL), a malignancy of TFH cells. Gene expression analyses of AITL showed that essentially all cases expressed elevated levels of transcripts for IL21, IL21R, and a series of genes associated with TFH cell development and function. These results identify a mouse model with features of AITL and suggest that patients with the disease might benefit from therapeutic interventions that interrupt IL-21 signaling.

Dissection of the neonatal Fc receptor (FcRn)-albumin interface using mutagenesis and anti-FcRn albumin-blocking antibodies.

Albumin is the most abundant protein in blood and plays a pivotal role as a multitransporter of a wide range of molecules such as fatty acids, metabolites, hormones, and toxins. In addition, it binds a variety of drugs. Its role as distributor is supported by its extraordinary serum half-life of 3 weeks. This is related to its size and binding to the cellular receptor FcRn, which rescues albumin from intracellular degradation. Furthermore, the long half-life has fostered a great and increasing interest in utilization of albumin as a carrier of protein therapeutics and chemical drugs. However, to fully understand how FcRn acts as a regulator of albumin homeostasis and to take advantage of the FcRn-albumin interaction in drug design, the interaction interface needs to be dissected. Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to unmask the binding sites for albumin blocking antibodies and albumin on the receptor, which revealed that the interaction is not only strictly pH-dependent, but predominantly hydrophobic in nature. Specifically, we provide mechanistic evidence for a crucial role of a cluster of conserved tryptophan residues that expose a pH-sensitive loop of FcRn, and identify structural differences in proximity to these hot spot residues that explain divergent cross-species binding properties of FcRn. Our findings expand our knowledge of how FcRn is controlling albumin homeostasis at a molecular level, which will guide design and engineering of novel albumin variants with altered transport properties.

IL-7/anti-IL-7 mAb complexes augment cytokine potency in mice through association with IgG-Fc and by competition with IL-7R.

Interleukin-7 (IL-7) is essential to T-cell survival as well as homeostatic proliferation, and clinical trials that exploit the mitogenic effects of IL-7 have achieved success in treating human diseases. In mice, the in vivo potency of IL-7 improves dramatically when it is administered as a complex with the anti-IL-7 neutralizing monoclonal antibody clone M25. However, the mechanism whereby M25 augments IL-7 potency is unknown. We have analyzed the discrete contributions of the antibody constant (Fc) and IL-7-binding (Fab) domains to the mechanism. By engaging the neonatal Fc receptor the Fc domain extends the in vivo lifespan of IL-7/M25 complexes and accounts for the majority of their activity. Unexpectedly, the IL-7-neutralizing Fab domain provides an additional, albeit smaller, contribution, possibly by serving as a cytokine depot. This study is the first to demonstrate that the neutralizing aspect of the monoclonal antibody is directly involved in enhancing the potency of a cytokine with a single form of receptor. Lessons from the mechanism of IL-7/M25 complexes inform the design of next-generation cytokine therapeutics.

IL-21 is a double-edged sword in the systemic lupus erythematosus-like disease of BXSB.Yaa mice.

The pleiotropic cytokine IL-21 is implicated in the pathogenesis of human systemic lupus erythematosus by polymorphisms in the molecule and its receptor (IL-21R). The systemic lupus erythematosus-like autoimmune disease of BXSB.Yaa mice is critically dependent on IL-21 signaling, providing a model for understanding IL-21/IL-21R signaling in lupus pathogenesis. In this study, we generated BXSB.Yaa mice selectively deficient in IL-21R on B cells, on all T cells, or on CD8(+) T cells alone and examined the effects on disease. We found that IL-21 signaling to B cells is essential for the development of all classical disease manifestations, but that IL-21 signaling also supports the expansion of central memory, CD8(+) suppressor cells and broadly represses the cytokine activity of CD4(+) T cells. These results indicate that IL-21 has both disease-promoting and disease-suppressive effects in the autoimmune disease of BXSB.Yaa mice.

Humanized FcRn mouse models for evaluating pharmacokinetics of human IgG antibodies.

A key element for the successful development of novel therapeutic antibodies is to fully understand their pharmacokinetic and pharmacodynamic behavior before performing clinical trials. While many in vitro modeling approaches exist, these simply cannot substitute for data obtained from appropriate animal models. It was established quite early that the unusual long serum half-life of immunoglobulin G's (IgGs) and Fc domains are due to their rescue and recycling by the neonatal Fc receptor (FcRn). The diverse roles of FcRn became apparent after isolation and cloning. Interesting are the significant species differences between rodent and human FcRn reactivity, rendering wild type rodents an inadequate model for studying IgG serum half-life. With the advance of genetic engineering mouse models have been established expressing human FcRn, and lacking mouse FcRn protein. These models have become highly relevant tools for serum half-life analysis of Fc-containing compounds.

Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection.

IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization using herpes simplex virus-2 (HSV-2)-specific polyclonal serum, which conferred significantly higher protection against intravaginal challenge infection by the HSV-2 186 strain in WT mice than in FcRn-KO mice. These studies demonstrate that FcRn-mediated transport is a mechanism by which IgG can act locally in the female genital tract in immune surveillance and in host defense against sexually transmitted diseases.

CD8+ T regulatory cells express the Ly49 Class I MHC receptor and are defective in autoimmune prone B6-Yaa mice.

The immune system includes a subpopulation of CD8(+) T cells equipped to inhibit the expansion of follicular T helper (T(FH)) cells, resulting in suppression of autoantibody production and associated lupus-like disease. These CD8(+) T regulatory (Treg) cells recognize Qa-1/peptide complexes on target T(FH) cells and depend on the IL-15 cytokine for development and function. Here we show that these CD8(+) Treg cells express a triad of surface receptors--CD44, CD122, and the class I MHC receptor Ly49--and account for <5% of CD8(+) T cells. Moreover, the development of systemic lupus erythematosus-like disease in B6-Yaa mutant mice is associated with a pronounced defect in CD8(+) Treg cell activity, suggesting that this regulatory subset may represent an effective therapeutic approach to systemic lupus erythematosus-like autoimmune disease.

Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport.

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin-Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG.

Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse.

The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.

Transcriptome analysis reveals organ-specific effects of 2-deoxyglucose treatment in healthy mice.

OBJECTIVE: Glycolytic inhibition via 2-deoxy-D-glucose (2DG) has potential therapeutic benefits for a range of diseases, including cancer, epilepsy, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and COVID-19, but the systemic effects of 2DG on gene function across different tissues are unclear. METHODS: This study analyzed the transcriptional profiles of nine tissues from C57BL/6J mice treated with 2DG to understand how it modulates pathways systemically. Principal component analysis (PCA), weighted gene co-network analysis (WGCNA), analysis of variance, and pathway analysis were all performed to identify modules altered by 2DG treatment. RESULTS: PCA revealed that samples clustered predominantly by tissue, suggesting that 2DG affects each tissue uniquely. Unsupervised clustering and WGCNA revealed six distinct tissue-specific modules significantly affected by 2DG, each with unique key pathways and genes. 2DG predominantly affected mitochondrial metabolism in the heart, while in the small intestine, it affected immunological pathways. CONCLUSIONS: These findings suggest that 2DG has a systemic impact that varies across organs, potentially affecting multiple pathways and functions. The study provides insights into the potential therapeutic benefits of 2DG across different diseases and highlights the importance of understanding its systemic effects for future research and clinical applications.

Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life.

Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood clearance in both strains of mice correlated with the hierarchy of binding obtained using soluble FcRn. Consequently, in vitro interaction analysis of engineered IgGs regarding their cross-species FcRn binding ability provides information for prediction of in vivo pharmacokinetics.

The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells.

The FcγRs found on macrophages (Ms) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Ms and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8(+) T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4(+) T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Ms but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4(+) T cells in the endosomal versus phagosomal compartments of Ms versus DCs.