Oral Cladribine Impairs Intermediate, but Not Conventional, Monocyte Transmigration in Multiple Sclerosis Patients across a Model Blood-Brain Barrier.

Multiple sclerosis (MS) is a disease in which the immune system damages components of the central nervous system (CNS), leading to the destruction of myelin and the formation of demyelinating plaques. This often occurs in episodic "attacks" precipitated by the transmigration of leukocytes across the blood-brain barrier (BBB), and repeated episodes of demyelination lead to substantial losses of axons within and removed from plaques, ultimately leading to progressive neurological dysfunction. Within leukocyte populations, macrophages and T and B lymphocytes are the predominant effectors. Among current immunotherapies, oral cladribine's impact on lymphocytes is well characterised, but little is known about its impact on other leukocytes such as monocytes and dendritic cells (DCs). The aim of this study was to determine the transmigratory ability of monocyte and DC subsets in healthy subjects and untreated and cladribine-treated relapse-remitting MS (RRMS) patients using a well-characterised model of the BBB. Peripheral blood mononuclear cells from subjects were added to an in vitro transmigration assay to assess cell migration. Our findings show that while prior treatment with oral cladribine inhibits the migration of intermediate monocytes, it has no impact on the transmigration of DC subsets. Overall, our data indicate a previously unrecognised role of cladribine on intermediate monocytes, known to accumulate in the brain active MS lesions.

Circulating CCR6+ILC proportions are lower in multiple sclerosis patients.

Objectives: The role of innate lymphoid cells (ILC), particularly helper ILC, in the pathogenesis of multiple sclerosis (MS) is not well understood. Here, we present a comprehensive analysis of peripheral ILC subsets in MS patients prior and after alemtuzumab administration using mass cytometry. Methods: Circulating ILC were analysed by mass cytometry in MS patients before and after alemtuzumab. These were compared with non-MS controls. MS-related shifts among ILC immunophenotypes were further elucidated by fast interpolation-based t-SNE (Flt-SNE) dimensionality reduction. Results: Neither natural killer (NK) cells nor helper ILC (ILC1, ILC2 and ILC3) levels were altered following alemtuzumab treatment. However, CD56bright NK cell expansions were observed in relapsing patients. MS patients prior to alemtuzumab further displayed proportional shifts from ILC1 to ILC2, with MS-associated decreases in CCR6+ helper ILC proportions. Conclusion: CD56bright NK cells during relapse indicate an immediate response to disease reactivation, while CCR6-related shifts among helper ILC suggest altered ILC migration to the CNS during MS.

Trans-Endothelial Migration of Memory T Cells Is Impaired in Alemtuzumab-Treated Multiple Sclerosis Patients.

The breakdown of the blood-brain barrier (BBB) and the trans-endothelial migration of lymphocytes are central events in the development of multiple sclerosis (MS). Autoreactive T cells are major players in MS pathogenesis, which are rapidly depleted following alemtuzumab treatment. This modulation, in turn, inhibits CNS inflammation, but alemtuzumab's effect on T cell migration into the CNS has been less studied. Human brain endothelial cells were stimulated with pro-inflammatory cytokines to mimic an inflamed BBB in vitro. Peripheral blood mononuclear cells from healthy controls, untreated or alemtuzumab-treated patients with relapsing-remitting MS (RRMS) were added to the BBB model to assess their transmigratory capacity. Here, the migration of CD4+ effector memory T (TEM) and CD8+ central memory T (TCM) cells across the BBB was impaired in alemtuzumab-treated patients. Naïve T (Tnaïve) cells were unable to migrate across all groups. CD38 was lowly expressed on CD8+ TCM cells, particularly for RRMS patients, compared to CD8+ Tnaïve cells. CD62L expression was lower on CD4+ TEM cells than CD4+ Tnaïve cells and decreased further in alemtuzumab-treated patients. These data suggest that repopulated memory T cells are phenotypically different from naïve T cells, which may affect their transmigration across the BBB in vitro.

Cladribine Reduces Trans-Endothelial Migration of Memory T Cells across an In Vitro Blood-Brain Barrier.

Multiple sclerosis (MS) is a chronic, demyelinating disease of the central nervous system (CNS) induced by immune dysregulation. Cladribine has been championed for its clinical efficacy with relatively minor side effects in treating MS. Although it is proposed that cladribine exerts an anti-migratory effect on lymphocytes at the blood-brain barrier (BBB) in addition to its lymphocyte-depleting and modulating effects, this has not been properly studied. Here, we aimed to determine if cladribine treatment influences trans-endothelial migration of T cell subsets across an inflamed BBB. Human brain endothelial cells stimulated with pro-inflammatory cytokines were used to mimic the BBB. Peripheral blood mononuclear cells were obtained from healthy controls, untreated and cladribine-treated MS patients. The trans-endothelial migration of CD4+ effector memory T (TEM) and CD8+ central memory T (TCM) cells was reduced in cladribine-treated MS patients. CD28 expression was decreased on both CD4+ TEM and CD8+ TCM cells, suggesting lowered peripheral activation of these cells thereby maintaining the integrity of the BBB. In addition, these cells have likely reconstituted following cladribine treatment, revealing a long-term anti-migratory effect. These results highlight new mechanisms by which cladribine acts to control MS pathogenesis.

Peripheral B-cell dysregulation is associated with relapse after long-term quiescence in patients with multiple sclerosis.

B cells play a major role in multiple sclerosis (MS), with many successful therapeutics capable of removing them from circulation. One such therapy, alemtuzumab, is thought to reset the immune system without the need for ongoing therapy in a proportion of patients. The exact cells contributing to disease pathogenesis and quiescence remain to be identified. We utilized mass cytometry to analyze B cells from the blood of patients with relapse-remitting MS (RRMS) before and after alemtuzumab treatment, and during relapse. A complementary RRMS cohort was analyzed by single-cell RNA sequencing. The R package "Spectre" was used to analyze these data, incorporating FlowSOM clustering, sparse partial least squares-discriminant analysis and permutational multivariate analysis of variance. Immunoglobulin (Ig)A+ and IgG1 + B-cell numbers were altered, including higher IgG1 + B cells during relapse. B-cell linker protein (BLNK), CD40 and CD210 expression by B cells was lower in patients with RRMS compared with non-MS controls, with similar results at the transcriptomic level. Finally, alemtuzumab restored BLNK, CD40 and CD210 expression by IgA+ and IgG1 + B cells, which was altered again during relapse. These data suggest that impairment of IgA+ and IgG1 + B cells may contribute to MS pathogenesis, which can be restored by alemtuzumab.

Selective modulation of trans-endothelial migration of lymphocyte subsets in multiple sclerosis patients under fingolimod treatment.

Multiple sclerosis (MS) is an autoimmune disorder where auto-aggressive T cells target the central nervous system (CNS), causing demyelination. The trans-endothelial migration of leucocytes across the blood-brain barrier (BBB) is one of the earliest CNS events in MS pathogenesis. We examined the effect of the disease state and treatment with fingolimod on the transmigration of peripheral blood mononuclear cells (PBMCs) in an in vitro BBB model. Patients' leucocyte numbers, subsets and phenotypes were assessed by flow cytometry. As expected, fingolimod treatment induced a significant reduction in T cell and B cell numbers compared to untreated MS patients and healthy controls. Interestingly fingolimod led to a marked reduction of CD4+ and a significant increase in CD8+ cell numbers. In migrated cells, only CD3+ cell numbers were reduced in fingolimod-treated, compared to untreated patients; it had no effect on B cell or monocyte transmigration. T cells were then differentiated into naïve, effector and memory subsets based on their expression of CCR7. This showed that MS patients had increased numbers of effector memory CD4+ cells re-expressing CD45RA (TEMRA) and a decrease in central memory (CM) CD8+ cells. The former was corrected by fingolimod, while the latter was not. CM CD4+ and CD8+ cells migrated across BBB more efficiently in fingolimod-treated patients. We found that while fingolimod reduced the proportions of naïve CD19+ B cells, it significantly increased the proportions of these cells which migrated. When B cells were further stratified based on CD24, CD27 and CD38 expression, the only effect of fingolimod was an enhancement of CD24hiCD27+ B cell migration, compared to untreated MS patients. The migratory capacities of CD8hi Natural Killer (NK), CD8dim NK and NK-T cells were also reduced by fingolimod. While the disease-modifying effects of fingolimod are currently explained by its effect on reducing circulating auto-aggressive lymphocytes, our data suggests that fingolimod may also have a direct though differential effect on the trans-endothelial migration of circulating lymphocyte populations.

IgG3 + B cells are associated with the development of multiple sclerosis.

OBJECTIVES: Disease-modifying therapies (DMTs) targeting B cells are amongst the most effective for preventing multiple sclerosis (MS) progression. IgG3 antibodies and their uncharacterised B-cell clones are predicted to play a pathogenic role in MS. Identifying subsets of IgG3 + B cells involved in MS progression could improve diagnosis, could inform timely disease intervention and may lead to new DMTs that target B cells more specifically. METHODS: We designed a 31-parameter B-cell-focused mass cytometry panel to interrogate the role of peripheral blood IgG3 + B cells in MS progression of two different patient cohorts: one to investigate the B-cell subsets involved in conversion from clinically isolated syndrome (CIS) to MS; and another to compare MS patients with inactive or active stages of disease. Each independent cohort included a group of non-MS controls. RESULTS: Nine distinct CD20+IgD-IgG3 + B-cell subsets were identified. Significant changes in the proportion of CD21+CD24+CD27-CD38- and CD27+CD38hiCD71hi memory B-cell subsets correlated with changes in serum IgG3 levels and time to conversion from CIS to MS. The same CD38- double-negative B-cell subset was significantly elevated in MS patients with active forms of the disease. A third CD21+CD24+CD27+CD38- subset was elevated in patients with active MS, whilst narrowband UVB significantly reduced the proportion of this switched-memory B-cell subset. CONCLUSION: We have identified previously uncharacterised subsets of IgG3 + B cells and shown them to correlate with autoimmune attacks on the central nervous system (CNS). These results highlight the potential for therapies that specifically target IgG3 + B cells to impact MS progression.

The Early Innate Immune Response to, and Phagocyte-Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis.

The innate immune system is the primary defense against cryptococcal infection, but paradoxically it promotes infection of the central nervous system. We performed a detailed longitudinal study of neurocryptococcosis in normal, chimeric, green fluorescent protein phagocyte-positive mice and phagocyte-depleted mice and interrogated the central nervous system innate immune response to Cryptococcus neoformans H99 using confocal microscopy, histology, flow cytometry, and quantification of brain cytokine/chemokines and fungal burdens. C. neoformans was present in the perivascular space (PVS) of post-capillary venules. This was associated with a massive influx of blood-derived monocytes, neutrophils, and T lymphocytes into the PVS and a predominantly proinflammatory cytokine/chemokine response. Phagocytes containing cryptococci were present only in the lumen and corresponding PVS of post-capillary venules. Free cryptococci were observed breaching the glia limitans, the protective barrier between the PVS and the cerebral parenchyma. Parenchymal cryptococcomas were typically in direct contact with post-capillary venules and lacked surrounding immune cell infiltrates. Phagocyte depletion abrogated cryptococcoma formation and PVS infiltrates. Together, these observations suggest that cryptococcomas can originate via phagocyte-dependent transport across post-capillary venular endothelium into the PVS and thence via passage of free cryptococci into the brain. In conclusion, we demonstrate for the first time that the PVS of cortical post-capillary venules is the major site of the early innate immune response to, and phagocyte-dependent entry of, C. neoformans.

Pho4 Is Essential for Dissemination of Cryptococcus neoformans to the Host Brain by Promoting Phosphate Uptake and Growth at Alkaline pH.

Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway. Cryptococcus neoformans disseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain (hlh3Δ/pho4Δ) defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, Hlh3/Pho4 functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. The pho4Δ mutant strain was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH, and under these conditions, the pho4Δ mutant had a growth defect and compromised phosphate uptake. The pho4Δ mutant was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. The pho4Δ mutant was not detected in blood, nor did it proliferate significantly when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on cryptococcal phosphate uptake and stress tolerance at alkaline pH, both of which are Pho4 dependent. IMPORTANCE Cryptococcal meningitis is fatal without treatment and responsible for more than 500,000 deaths annually. To be a successful pathogen, C. neoformans must obtain an adequate supply of essential nutrients, including phosphate, from various host niches. Phosphate acquisition in fungi is regulated by the PHO signaling cascade, which is activated when intracellular phosphate decreases below a critical level. Induction of phosphate acquisition genes leads to the uptake of free phosphate via transporters. By blocking the PHO pathway using a Pho4 transcription factor mutant (pho4Δ mutant), we demonstrate the importance of the pathway for cryptococcal dissemination and the establishment of brain infection in murine models. Specifically, we show that reduced dissemination of the pho4Δ mutant to the brain is due to an alkaline pH tolerance defect, as alkaline pH mimics the conditions of phosphate deprivation. The end result is inhibited proliferation in host tissues, particularly in blood.

Cryptococcal transmigration across a model brain blood-barrier: evidence of the Trojan horse mechanism and differences between Cryptococcus neoformans var. grubii strain H99 and Cryptococcus gattii strain R265.

Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause neurological disease and cross the BBB as free cells or in mononuclear phagocytes via the Trojan horse mechanism, although evidence for the latter is indirect. There is emerging evidence that Cn and the North American outbreak Cg strain (R265) more commonly cause neurological and lung disease, respectively. We have employed a widely validated in vitro model of the BBB, which utilizes the hCMEC/D3 cell line derived from human brain endothelial cells (HBEC) and the human macrophage-like cell line, THP-1, to investigate whether transport of dual fluorescence-labelled Cn and Cg across the BBB occurs within macrophages. We showed that phagocytosis of Cn by non-interferon (IFN)-γ stimulated THP-1 cells was higher than that of Cg. Although Cn and Cg-loaded THP-1 bound similarly to TNF-activated HBECs under shear stress, more Cn-loaded macrophages were transported across an intact HBEC monolayer, consistent with the predilection of Cn for CNS infection. Furthermore, Cn exhibited a higher rate of expulsion from transmigrated THP-1 compared with Cg. Our results therefore provide further evidence for transmigration of both Cn and Cg via the Trojan horse mechanism and a potential explanation for the predilection of Cn to cause CNS infection.

Discovery of potent, selective, and orally bioavailable alkynylphenoxyacetic acid CRTH2 (DP2) receptor antagonists for the treatment of allergic inflammatory diseases.

New phenoxyacetic acid antagonists of CRTH2 are described. Following the discovery of a hit compound by a focused screening, high protein binding was identified as its main weakness. Optimization aimed at reducing serum protein binding led to the identification of several compounds that showed not only excellent affinities for the receptor (41 compounds with K(i) < 10 nM) but also excellent potencies in a human whole blood assay (IC(50) < 100 nM; PGD2-induced eosinophil shape change). Additional optimization of the PK characteristics led to the identification of several compounds suitable for in vivo testing. Of these, 19k and 19s were tested in two different pharmacological models (acute FITC-mediated contact hypersensitivity and ovalbumin-induced eosinophilia models) and found to be active after oral dosing (10 and 30 mg/kg).

GLEPP1/protein-tyrosine phosphatase phi inhibitors block chemotaxis in vitro and in vivo and improve murine ulcerative colitis.

We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase . Two previously described GLEPP1 substrates, paxillin and Syk, are both required for cytoskeletal rearrangement and cellular motility of leukocytes in chemotaxis. We show here that GLEPP1 inhibitors prevent dephosphorylation of Syk1 and paxillin in resting cells and block primary human monocyte and mouse bone marrow-derived macrophage chemotaxis in a gradient of monocyte chemotactic protein-1. In mice, the GLEPP1 inhibitors also reduce thioglycolate-induced peritoneal chemotaxis of neutrophils, lymphocytes, and macrophages. In murine disease models, the GLEPP1 inhibitors significantly reduce severity of contact hypersensitivity, a model for allergic dermatitis, and dextran sulfate sodium-induced ulcerative colitis, a model for inflammatory bowel disease. Taken together, our data provide confirmation that GLEPP1 plays an important role in controlling chemotaxis of multiple types of leukocytes and that pharmacological inhibition of this phosphatase may have therapeutic use.

Inhibition of airway inflammation by amino-terminally modified RANTES/CC chemokine ligand 5 analogues is not mediated through CCR3.

Chemokines play a key role in the recruitment of activated CD4(+) T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 micro g of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1beta/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.

IL-18-independent cytotoxic T lymphocyte activation and IFN-gamma production during experimental acute graft-versus-host disease.

Acute graft-versus-host disease (GvHD) is a serious complication after allogeneic bone marrow transplantation. Donor-derived T cells infiltrate recipient target organs and cause severe tissue damage, often leading to death of the affected patient. Tissue destruction is a direct result of donor CD8+ T cell activation and cell-mediated cytotoxicity. IL-18 is a novel pro-inflammatory cytokine with potent T(h)1 immune response-promoting and cytotoxic T lymphocyte (CTL)-inducing activity. IL-18 is strongly induced in experimental mouse models and human patients with acute GvHD. However, the precise role of IL-18 in the development of acute GvHD is still unknown. In this study, we have used IL-18-binding protein, a soluble IL-18 decoy receptor, to specifically neutralize IL-18 in vivo and in vitro. Our results demonstrate that IL-18 is induced during GvHD. However, its effect in the induction of GvHD appears to be redundant, since neutralization of IL-18 does not alter any disease parameter analyzed. Our study further shows that IFN-gamma production and CTL induction upon activation by T cell mitogens or by alloantigen does not involve IL-18-mediated amplification, in contrast to lipopolysaccharide-induced IFN-gamma production. We conclude that IL-18 expression correlates with the course of GvHD; however, its effect is dispensable for IFN-gamma and CTL induction for the initiation phase of this disease, most likely due to direct, IL-18-independent, CTL activation.