CD11c+CD88+CD317+ myeloid cells are critical mediators of persistent CNS autoimmunity.

Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c+ cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre+/-ITGA4fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c+ cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c+ cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre+/-ITGA4fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre+/-ITGA4fl/fl mice, during which CD11c+CD88+ cells were sequestered in the blood. Upon clinical EAE onset, CD11c+CD88+ cells appeared in the CNS and expressed CD317+ In adoptive transfer experiments, CD11c.Cre+/-ITGA4fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c+CD88+CD317+ cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

Limitations of cell-lineage-specific non-dynamic gene recombination in CD11c.Cre+ITGA4fl/fl mice.

BACKGROUND: The Cre-lox system is a non-dynamic method of gene modification and characterization. Promoters thought to be relatively cell-specific are utilized for generation of cell-lineage-specific gene modifications. METHODS: CD11c.Cre+ITGA4fl/fl mice were generated to abolish the expression of ITGA (α4-integrin) in CD11c+ cells. Ex vivo flow cytometry studies were used to assess the expression of cellular surface markers in different lymphoid compartments and leukocytes subsets after Cre-mediated recombination. RESULTS: A significant reduction of α4-integrin expression among CD11c+- cells was achieved in CD11c.Cre+ITGA4fl/fl mice in primary and secondary lymphoid tissues. A similar reduction in the expression of α4-integrin was also observed in CD11c- cells. CONCLUSION: Cre-lox-mediated cell lineage-specific gene deletion is limited by the transient expression of recombination regulating sequences in hematopoietic cell lines. These methodological issues indicate the need to consider when to employ non-dynamic DNA recombination models in animal models of CNS autoimmunity. An experimental algorithm to address the biological complexities of non-dynamic gene recombination is provided.

α4-integrin deficiency in B cells does not affect disease in a T-cell-mediated EAE disease model.

Objective: The goal of this study was to investigate the role of CD 19+ B cells within the brain and spinal cord during CNS autoimmunity in a peptide-induced, primarily T-cell-mediated experimental autoimmune encephalomyelitis (EAE) model of MS. We hypothesized that CD19+ B cells outside the CNS drive inflammation in EAE. Methods: We generated CD19.Cre+/- α4-integrinfl/fl mice. EAE was induced by active immunization with myelin oligodendrocyte glycoprotein peptide (MOGp35-55). Multiparameter flow cytometry was used to phenotype leukocyte subsets in primary and secondary lymphoid organs and the CNS. Serum cytokine levels and Ig levels were assessed by bead array. B-cell adoptive transfer was used to determine the compartment-specific pathogenic role of antigen-specific and non-antigen-specific B cells. Results: A genetic ablation of α4-integrin in CD19+/- B cells significantly reduced the number of CD19+ B cells in the CNS but does not affect EAE disease activity in active MOGp35-55-induced disease. The composition of B-cell subsets in the brain, primary lymphoid organs, and secondary lymphoid organs of CD19.Cre+/- α4-integrinfl/fl mice was unchanged during MOGp35-55-induced EAE. Adoptive transfer of purified CD19+ B cells from CD19.Cre+/- α4-integrinfl/fl mice or C57BL/6 wild-type (WT) control mice immunized with recombinant rMOG1-125 or ovalbumin323-339 into MOGp35-55-immunized CD19.Cre+/- α4-integrinfl/fl mice caused worse clinical EAE than was observed in MOGp35-55-immunized C57BL/6 WT control mice that did not receive adoptively transferred CD19+ B cells. Conclusions: Observations made in CD19.Cre+/- α4-integrinfl/fl mice in active MOGp35-55-induced EAE suggest a compartment-specific pathogenic role of CD19+ B cells mostly outside of the CNS that is not necessarily antigen specific.

Defining standard enzymatic dissociation methods for individual brains and spinal cords in EAE.

OBJECTIVE: To determine the capacity, effectiveness, efficiency, and reliability of select tissue dissociation methods to isolate mononuclear cells from the CNS of mice with experimental autoimmune encephalomyelitis (EAE). METHODS: As part of an assay qualification, we tested the isolation method Percoll PLUS vs a commercially available enzymatic Neural Tissue Dissociation Kit (Kit), and the enzymes accutase and papain in C57BL/6 mice with active EAE. In a stepwise approach, we applied the following 4 criteria to each dissociation method: (1) mononuclear cell viability post-processing was required to be ≥80% per brain or spinal cord sample, (2) absolute live mononuclear cell numbers was required to be ≥5 × 105 per brain or spinal cord sample of mice with clinical EAE, (3) test-retest reliability had to be verified, and (4) the absolute mononuclear cell numbers in brain and spinal cord had to correlate with the EAE disease course. RESULTS: Enzymatic dissociations allowed for greatly increased cell yield and specifically allowed for downstream assays from individual brains and spinal cords in C57BL/6 mice with EAE. All enzymatic dissociations provided a more efficient and effective method for isolating mononuclear cells from brains and spinal cord. Only the Kit assay provided a significant correlation between absolute mononuclear cell numbers in the spinal cord and EAE disease severity. CONCLUSIONS: Enzymatic dissociation of CNS tissue of C57BL/6 mice with active EAE with the Kit should be the standard method. The identification of optimized CNS dissociation methods in EAE has the potential to identify cellular events that are pertinent to MS pathogenesis.

PDCB does not promote CNS autoimmunity in the context of genetic susceptibility but worsens its outcome.

BACKGROUND: Para-dichlorobenzene (PDCB) is an aromatic hydrocarbon contained in mothballs that is potentially neurotoxic. A potential pathogenic role of PDCB in MS pathogenesis has been suggested. METHODS: To determine the ability of chronic PDCB ingestion to induce CNS autoimmunity in a genetically susceptible mammalian species, naive myelin oligodendrocyte glycoprotein peptide (MOGp)35-55 T cell receptor (TCR) transgenic mice (2D2) on the C57Bl/6 background were orally gavaged once daily with corn oil control, 125 mg/kg PDCB, or 250 mg/kg PDCB for 45 days. The incidence of spontaneous EAE is increased in this mouse strain. RESULTS: Both PDCB treatment groups showed the same spontaneous incidence of EAE, an earlier disease onset, and a slight decrease in survival for 125 mg/kg PDCB mice compared to control mice. We were unable to detect any PDCB, or its metabolites 2,5-dichlorophenol, 2,5-dicholormethylsulfide, and 2,5-dichloromethylsulfone in the brain and spinal cord of control mice. In contrast, PDCB was readily detectable in both compartments in mice who received PDCB via oral gavage, with concentrations being significantly higher in the brain (p < 0.01). Levels of the metabolites 2,5-dichlorophenol and 2,5-dichloromethylsulfone were also significantly higher in brains compared to spinal cords. CONCLUSION: Our study refutes the hypothesis that PDCB or its metabolites trigger spontaneous T cell-mediated CNS autoimmunity in the setting of genetic susceptibility. A slight increase in mortality with PDCB exposure may be due systemic toxicity of hydrocarbons.

TLR3 agonism re-establishes CNS immune competence during α4-integrin deficiency.

OBJECTIVE: Natalizumab blocks α4-integrin-mediated leukocyte migration into the central nervous system (CNS). It diminishes disease activity in multiple sclerosis (MS), but carries a high risk of progressive multifocal encephalopathy (PML), an opportunistic infection with JV virus that may be prompted by diminished CNS immune surveillance. The initial host response to viral infections entails the synthesis of type I interferons (IFN) upon engagement of TLR3 receptors. We hypothesized that TLR3 agonism reestablishes CNS immune competence in the setting of α4-integrin deficiency. METHOD: We generated the conditional knock out mouse strain Mx1.Cre+ α4-integrinfl/fl, in which the α4-integrin gene is ablated upon treatment with the TLR3 agonist poly I:C. Adoptive transfer of purified lymphocytes from poly I:C-treated Mx1.Cre+ α4-integrinfl/fl donors into naive recipients recapitulates immunosuppression under natalizumab. Active experimental autoimmune encephalomyelitis (EAE) in Mx1.Cre+ α4-integrinfl/fl mice treated with poly I:C represents immune-reconstitution. RESULTS: Adoptive transfer of T cells from poly I:C treated Mx1.Cre+ α4-integrinfl/fl mice causes minimal EAE. The in vitro migratory capability of CD45+ splenocytes from these mice is reduced. In contrast, actively-induced EAE after poly I:C treatment results in full disease susceptibility of Mx1.Cre+ α4-integrinfl/fl mice, and the number and composition of CNS leukocytes is similar to controls. Extravasation of Evans Blue indicates a compromised blood-brain barrier. Poly I:C treatment results in a 2-fold increase in IFN ß transcription in the spinal cord. INTERPRETATION: Our data suggest that TLR3 agonism in the setting of relative α4-integrin deficiency can reestablish CNS immune surveillance in an experimental model. This pathway may present a feasible treatment strategy to treat and prevent PML under natalizumab therapy and should be considered for further experimental evaluation in a controlled setting.

Microbial co-infection alters macrophage polarization, phagosomal escape, and microbial killing.

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guerin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24-48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection.