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Fatigue is the most frequent symptom in multiple sclerosis (MS), although it is still poorly understood due to its complexity and subjective nature. There is an urgent need to identify reliable biomarkers to improve disease prognosis and therapeutic strategies. Epstein-Barr virus (EBV) is the major environmental risk factor associated with MS aetiology, and trials with EBV-targeted T cell therapies have reduced fatigue severity in MS patients.We investigated whether the serum amount of immunoglobulin (Ig)G-specific for EBV antigens could be a suitable prognostic marker for the assessment of MS-related fatigue.A total of 194 MS patients were enrolled. We quantified EBV nuclear antigen 1 (EBNA1) and EBV viral capsid antigen (VCA) immunoglobulin (Ig) G levels and B cell-activating factor of the tumour necrosis factor family (BAFF) concentration in the serum of patients with relapsing-remitting MS (RRMS) and chronic progressive MS (CPMS), and we analysed their correlation with aspects of fatigue and other clinical disease parameters.A complete EBV seropositivity could be detected in our cohort. After adjusting for confounding variables and covariates, neither EBNA1 nor VCA antibody titres were associated with levels of fatigue, sleepiness, depression, or with any of the clinical values such as expanded disability status scale, lesion count, annual relapse rate, or disease duration. However, patients with RRMS had significantly higher EBNA1 IgG titre than those with CPMS, whereas this was not the case under therapies targeting CD20+ cells. BAFF levels in serum were inversely proportional to anti-EBNA1 IgG.Our results show that EBNA1 IgG titre is not associated with the presence or level of fatigue. Whether the increased EBNA1 titre in RRMS plays a direct role in disease progression, or is only a consequence of excessive B cell activation, remains to be answered in future studies.

Background: Oral cladribine is an approved disease-modifying drug for the treatment of relapsing multiple sclerosis. In controlled clinical trials as well as in post marketing safety assessments, autoimmune conditions have not yet been reported as a specific side effect of cladribine. Objective and Results: Here, we report a case of anti-glomerular basement membrane antibody-mediated glomerulonephritis that occurred shortly after the fourth cladribine treatment cycle. Conclusion: Neurologists should be attentive to the development of secondary autoimmunity in cladribine-treated patients.

IL-17 is associated with different phenotypes of asthma, however, it is not fully elucidated how it influences induction and maintenance of asthma and allergy. In order to determine the role of IL-17 in development of allergic asthma, we used IL-17A/F double KO (IL-17A/F KO) and WT mice with or without neutralization of IL-17 in an experimental allergic asthma model and analyzed airway hyperresponsiveness, lung inflammation, T helper cell polarization, and DCs influx and activation. We report that the absence of IL-17 reduced influx of DCs into lungs and lung draining LNs. Compared to WT mice, IL-17A/F KO mice or WT mice after neutralization of IL-17A showed reduced airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and IgE levels. DCs from draining LNs of allergen-challenged IL-17A/F KO mice showed a reduction in expression of migratory and costimulatory molecules CCR7, CCR2, MHC-II, and CD40 compared to WT DCs. Moreover, in vivo stimulation of adoptively transferred antigen-specific cells was attenuated in lung-draining LNs in the absence of IL-17. Thus, we report that IL-17 enhances airway DC activation, migration, and function. Consequently, lack of IL-17 leads to reduced antigen-specific T cell priming and impaired development of experimental allergic asthma.

Background: Cladribine is a synthetic deoxyadenosine analogue approved for the treatment of highly active relapsing multiple sclerosis (RMS). Cladribine is considered to be a semi-selective immune-reconstitution therapy (IRT) that induces long-term remission following short course of treatment. Here, we evaluated the effect of cladribine on immune cell reduction and reconstitution during the first two years of treatment. Methods: We analyzed our longitudinal, prospective, real-world cohort of 80 cladribine-treated RMS patients from two tertiary centers in Germany. Laboratory testing was conducted monthly and included evaluation of cellular as well as soluble parameters. Laboratory outcomes were correlated with infectious adverse events (AEs) and clinical or paraclinical disease activity. Results: Selective alterations in immune cell populations occurred following cladribine treatment, with the most marked effects observed in year two of treatment. Specifically, a rapid reduction in CD56+ natural killer cells (nadir: month 1 (year 1) and 14 (year 2); -37 and -41% from baseline) was followed by a greater reduction in CD19+ B cells (nadir: month 2 and 14; -81 and -82%); a moderate effect on CD4+ (nadir: month 3 and 15; -48 and -61%) and CD8+ T cells (nadir: month 5 and 18; -40 and -48%). Despite the marked effect on B cells, immunoglobulin levels were unaffected. There was no or minimal effect on thrombocytes and innate immune cells. Clinical and paraclinical disease activity was unrelated to the observed immune alterations. Lymphopenia was the most commonly observed AE (86.3% of patients; grade III-IV lymphopenia: 38.8%). The cumulative incidence of infections was 55% with cladribine treatment, which were mostly mild or moderate. In total, 19 herpes infections developed in 8 (10%) cladribine-treated patients; all cases were dermatomal and 94.7% of the herpetic infections occurred during a period of lymphopenia. Conclusions: The immunophenotyping data obtained in our real-world setting are comparable to those demonstrated in pivotal clinical trials and provide further evidence that cladribine may represent a form of IRT. However, regarding the side-effect profile of cladribine, severe lymphopenia (exceeding grade II CTCAE) was more frequent, which may have prompted the development of herpes infections. Of note, lymphocyte dynamics did not correlate with clinical and paraclinical measures of disease activity in the two-year follow-up period.

Sepsis predisposes for poor stroke outcome. This association suggests that sepsis disturbs post-ischemic tissue survival and brain remodeling. To elucidate this link, we herein exposed mice to 30 min intraluminal middle cerebral artery occlusion (MCAO) and induced a sepsis-like state at 72 h post-ischemia by intraperitoneal delivery of Escherichia coli lipopolysaccharide (LPS; three doses of 0.1 or 1 mg/kg, separated by 6 h), a major component of the bacterium’s outer membrane. Neurological recovery, ischemic injury, brain remodeling and immune responses were evaluated over up to 56 days post-sepsis (dps) by behavioral tests, immunohistochemistry and flow cytometry. Delivery of 1 mg/kg but not 0.1 mg/kg LPS reduced rectal temperature over 48 h by up to 3.4 ± 3.1 °C, increased general and focal neurological deficits in the Clark score over 72 h and increased motor-coordination deficits in the tight rope test over up to 21 days. Notably, 1 mg/kg, but not 0.1 mg/kg LPS increased intercellular adhesion molecule-1 abundance on ischemic microvessels, increased microvascular thrombosis and increased patrolling monocyte and T cell infiltrates in ischemic brain tissue at 3 dps. Infarct volume was increased by 1 mg/kg, but not 0.1 mg/kg LPS at 3 dps (that is, 6 days post-MCAO), as was brain atrophy at 28 and 56 dps. Microglial activation in ischemic brain tissue, evaluated by morphology analysis of Iba-1 immunostainings, was transiently increased by 0.1 and 1 mg/kg LPS at 3 dps. Our data provide evidence that neurological recovery and brain remodeling are profoundly compromised in the ischemic brain post-sepsis as a consequence of cerebral thromboinflammation.

Background and Purpose— Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods— Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV–treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results— sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O 2 ) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O 2 ) but also reduced serum IgG extravasation—a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions— Leukocytes and specifically PMNs contribute to MSC-sEV–induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview— An online visual overview is available for this article.

To study and isolate lung cells by flow cytometry, enzymatic digestion and generation of single cell suspensions is required. This significantly influences expression of cellular epitopes and protocols need to be adapted for the best isolation and subsequent analysis of specific cellular subsets.We optimized protocols for the simultaneous isolation and characterization of specific human and murine lung cell types. For alveolar epithelial cells (AEC), a primarily dispase based digestion method and for leukocytes, a primarily collagenase based technique was adapted. Protocols were applied in parallel in either single experimental mice or human lung specimens.Optimized dispase/DNase digestion yielded a high percentage of Epcam+CD45-CD31- AEC as assessed by flow cytometry. Epcam+CD45-CD3-CD11b-CD11c-CD16/32-CD19-CD31-F4/80- AEC were readily sortable with high purity and typical morphology and function upon in vitro stimulation with lipopolysaccharide or respiratory-syncytial-virus (RSV) infection. To analyze lung leukocytes, specimens were digested with an adapted collagenase/DNase protocol yielding high percentages of viable leukocytes with typical morphology, function, and preserved subset specific leukocyte markers. Both protocols could be applied simultaneously in a single experimental mouse post mortem. Application of both digestion methods in primary human lung specimens yielded similar results with high proportions of Epcam+CD45- human AEC after dispase/DNase digestion and preservation of human T cell epitopes after collagenase/DNase digestion.The here described protocols were optimized for the simple and efficient isolation of murine and human lung cells. In contrast to previously described techniques, they permit simultaneous in-depth characterization of pulmonary epithelial cells and leukocyte subsets such as T helper, cytotoxic T, and B cells from one sample. As such, they may help to comprehensively and sustainably characterize murine and human lung specimens and facilitate studies on the role of lung immune cells in different respiratory pathologies.

Background Asthma is a widespread, multifactorial chronic airway disease. The influence of regulatory B cells on airway hyperreactivity (AHR) and remodeling in asthma is poorly understood. Objective Our aim was to analyze the role of B cells in a house dust mite (HDM)-based murine asthma model. Methods The influence of B cells on lung function, tissue remodeling, and the immune response were analyzed by using wild-type and B-cell–deficient (μMT) mice and transfer of IL-10–proficient and IL-10–deficient B cells to μMT mice. Results After HDM-sensitization, both wild-type and μMT mice developed AHR, but the AHR was significantly stronger in μMT mice, as confirmed by 2 independent techniques: invasive lung function measurement in vivo and examination of precision-cut lung slices ex vivo. Moreover, airway remodeling was significantly increased in allergic μMT mice, as shown by enhanced collagen deposition in the airways, whereas the numbers of FoxP3+ and FoxP3– IL-10–secreting regulatory T cells were reduced. Adoptive transfer of IL-10–proficient but not IL-10–deficient B cells into μMT mice before HDM-sensitization attenuated AHR and lung remodeling. In contrast, FoxP3+ regulatory T cells were equally upregulated by transfer of IL-10–proficient and IL-10–deficient B cells. Conclusion Our data in a murine asthma model illustrate a central role of regulatory B cells in the control of lung function and airway remodeling and may support future concepts for B-cell–targeted prevention and treatment strategies for allergic asthma.

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor β1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-β type 2 receptor expression, particularly in CD14+ cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guerin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria.

Allergic asthma is a widespread chronic inflammatory disease of the airways. The role of different B cell subsets in developing asthma and respiratory tolerance is not well known. Especially regulatory B (Breg) cells are proposed to be important in asthma regulation. Using wild-type (WT) and B cell-deficient (μMT) mice we investigated how B cells are affected by induction of allergic airway inflammation and respiratory tolerance and whether they are necessary to develop these conditions. WT mice with an asthma-like phenotype, characterized by increased airway hyper reactivity, eosinophilic airway inflammation, mucus hypersecretion and elevated Th2 cytokines, exhibited increased MHCII and CD23 expression on follicular mature B cells in lung, bronchial lymph nodes (bLN) and spleen, which contributed to allergen-specific T cell proliferation in vitro. Germinal center B cell numbers were elevated and associated with increased production of allergen-specific immunoglobulins especially in bLN. In contrast, respiratory tolerance clearly attenuated these B cell alterations and directly enhanced marginal zone precursor B cells, which induced regulatory T cells in vitro. However, μMT mice developed asthma-like and tolerized phenotypes like WT mice. Our data indicate that although B cell subsets are affected by asthma-like and respiratory tolerant phenotypes, B cells are not required for tolerance induction.

Different models of experimental allergic asthma have shown that the TLR7/8 agonist resiquimod (R848) is a potential inhibitor of type 2 helper cell–driven inflammatory responses. However, the mechanisms mediating its therapeutic effects are not fully understood. Using a model of experimental allergic asthma, we show that induction of IL-27 by R848 is critical for the observed ameliorative effects. R848 significantly inhibited all hallmarks of experimental allergic asthma, including airway hyperreactivity, eosinophilic airway inflammation, mucus hypersecretion, and Ag-specific Ig production. Whereas R848 significantly reduced IL-5, IL-13, and IL-17, it induced IFN-γ and IL-27. Neutralization of IL-27 completely reversed the therapeutic effect of R848 in the experimental asthma model, demonstrating dependence of R848-mediated suppression on IL-27. In vitro, R848 induced production of IL-27 by murine alveolar macrophages and dendritic cells and enhanced expression of programmed death–ligand 1, whose expression on monocytes and dendritic cells has been shown to regulate peripheral tolerance in both murine and human studies. Moreover, in vitro IL-27 enhanced secretion of IFN-γ whereas it inhibited IL-5 and IL-13, demonstrating its direct effect on attenuating Th2 responses. Taken together, our study proves that R848-mediated suppression of experimental asthma is dependent on IL-27. These data provide evidence of a central role of IL-27 for the control of Th2-mediated allergic diseases.

Asthma is a widespread chronic multifactorial disease of the airways which is often triggered by the perennial allergen house dust mite (HDM). Next to regulatory T cells (Tregs), IL10 producing regulatory B cells are crucial to substantially counteract inflammatory responses. However, their regulatory impact in asthma is inadequately known so far. To better understand the regulatory influence of B cells in allergic asthma, we employed a HDM-based experimental asthma model using wildtype (WT) and B cell deficient (µMT) mice. Compared to WT counterparts, HDM-sensitized µMT mice developed a significantly increased airway hyperreactivity (AHR), a cardinal feature of asthma, measured by invasive lung function. This observation was confirmed by independent precision cut lung slices (PCLS) technique, demonstrating stronger bronchoconstriction of explanted lung tissue slices of allergic µMT mice after methacholine provocation. Furthermore, allergic µMT mice displayed diminished levels of Tregs and Th2 cytokines, especially IL10, as determined by flow cytometry and immunoassays. Adoptive transfer of naive WT B cells followed by HDM-sensitization substantially attenuated AHR in µMT mice and boosted IL10 production and Treg expansion. To test the impact of IL10, B cells from IL10 deficient (IL10-/-) mice were transferred, but failed to improve lung function of allergic µMT mice. Furthermore, transfer of WT and IL10-/- B cells provoked comparable amounts of IL10 and Tregs. Taken together, our data indicate that B cells are critical in HDM-induced AHR, and that IL10 competent B cells may be central in regulating AHR in allergic airway inflammation.

Regulatory T cells (Treg) maintain immunological self-tolerance and their functional or numerical deficits are associated with progression of several neurological diseases. We examined the effects of Treg absence on the structure and integrity of the unchallenged murine brain. When compared to control, Treg-deficient FoxP3sf mutant mice showed no differences in brain size, myelin amount and oligodendrocyte numbers. FoxP3sf strain displayed no variations in quantity of neurons and astrocytes, whereas microglia numbers were slightly reduced. We demonstrate lack of neuroinflammation and parenchymal responses in the brains of Treg-deficient mice, suggesting a minor Treg role in absence of blood-brain barrier breakdown.

Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood.To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages.Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays.Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA.These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

Background Allergic asthma is a chronic lung disease resulting from inappropriate immune responses to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma. Objective We analyzed the mechanisms of perinatal tolerance induction to allergens, with particular focus on the role of B cells in preconception and early intrauterine immune priming. Methods Wild-type (WT) and B cell–deficient mice received ovalbumin (OVA) intranasally before mating. Their offspring were analyzed in a murine model of allergic airway inflammation. Results Although antigen application before conception protected WT progeny from allergy, it aggravated allergic airway inflammation in B cell–deficient offspring. B-cell transfer restored protection, demonstrating the crucial role of B cells in perinatal tolerance induction. Effective diaplacentar allergen transfer was detectable in pregnant WT mice but not in pregnant B-cell knockout dams, and antigen concentrations in WT amniotic fluid (AF) were higher than in IgG-free AF of B cell–deficient dams. Application of OVA/IgG immune complexes during pregnancy boosted OVA uptake by fetal dendritic cells (DCs). Fetal DCs in human subjects and mice expressed strikingly higher levels of Fcγ receptors compared with DCs from adults and were highly efficient in taking up OVA/IgG immune complexes. Moreover, murine fetal DCs effectively primed antigen-specific forkhead box P3 + regulatory T cells after in vitro coincubation with OVA/IgG-containing AF. Conclusion Our data support a decisive role for B cells and immunoglobulins during in utero tolerance priming. These findings improve the understanding of perinatal immunity and might support the development of effective primary prevention strategies for allergy and asthma in the future.

Microglia play a key role in the initiation and perpetuation of de- and remyelination because of their ability to present antigens and clear cell debris by phagocytosis. Different factors expressed or secreted by microglia seem to play an important role in regenerative processes. But it remains unclear which factors lead to a protective microglial phenotype and recent data indicate region-specific differences within the central nervous system (CNS) for both de-/remyelination and microglial response. In order to identify important factors that promote neuroprotection, we examined changes in microglial phenotypes in the cuprizone model. We undertook an extensive and detailed analysis of the expression of surface markers as well as cytokines, growth factors, and the phagocytosis activity of microglia. We found a pronounced increase of phagocytosis activity of microglia during demyelination associated with an upregulation of phagocytic receptors, from which TREM-2b was the most prominent. The expression of MHC II was only increased at the peak of demyelination but costimulatory molecules showed no significant changes. Interestingly, the proinflammatory cytokine TNF-α was upregulated while the anti-inflammatory cytokines IL-10 and TGF-ß remained unchanged. The growth factors IFG-1 and FGF-2, which were both suggested to promote remyelination, were increased during demyelination. Our findings characterise changes of microglial markers during de- and remyelination indicating that debris clearance mediated via TREM-2b plays a central role in the regulation of these processes. Microglial phagocytosis as well as production of TNF-α, IGF-1, and FGF-2 seems to be important factors for the creation of an environment promoting regeneration.

Systemic infection can influence the course in many diseases of the central nervous system (CNS) such as multiple sclerosis (MS), yet the relationship between infection outside the CNS and potential damage and/or protection within the CNS is still not understood. Activation of microglia is a characteristic feature of most CNS autoimmune disorders, including MS, and both protective and degenerative functions of microglia have been proposed. Hence, we analyzed the effects of a systemic inflammatory reaction induced by peripheral treatment with lipopolysaccharide (LPS) on microglial reaction and cuprizone induced de- and remyelination. We found that LPS administration delayed demyelination, which was linked with inhibition of microglial proliferation and reduced numbers of activated microglia. The phenotype of microglia changed as an increase of Toll-like receptor 4 was found. During remyelination, LPS treatment delayed the onset of myelin protein re-expression, but later there was a beneficial effect via an increase of proliferating oligodendrocyte precursor cells (OPC) and mature oligodendrocytes. Moreover, the expression of ciliary neurotrophic factor was increased in response to LPS, a growth factor known to mediate OPC proliferation. Additional experiments showed that the time window to induce LPS effects was limited and associated with the presence of microglia. In conclusion, LPS delayed demyelination and caused beneficial effects on remyelination via increasing the proliferation of OPC. These differences seem to be an effect of LPS induced microglial modulation and indicate that exposure to certain infectious agents within a given time window may be beneficial in promoting tissue repair.

Apart from their involvement in the pathogenesis of demyelinating diseases such as multiple sclerosis, there is emerging evidence that matrix metalloproteinases (MMPs) also promote remyelination. We investigated region-specific expression patterns of 11 MMPs and 4 tissueinhibitors of metalloproteinases (TIMPs) in the cuprizone murine demyelination model. Messenger RNA (mRNA) was extracted at different time points of exposure to cuprizone from microdissected samples of corpus callosum, cortex, and ex vivo isolated microglia and analyzedusing quantitative reverse transcription-polymerase chain reaction.Matrix metalloproteinase 12 and TIMP-1 mRNA were significantly upregulated versus age-matched controls in both areas during demyelination and remyelination. Matrix metalloproteinases 3, 11, and 14 mRNA were upregulated only in white matter during remyelination. Matrix metalloproteinase 24 mRNA was downregulated during both demyelination and remyelination. To identify potential cellular sources of the MMPs and TIMPs, we isolated microglia and detected high MMP-12and TIMP-2 mRNA upregulation at the peak of demyelination.By immunohistochemistry, MMP-3 protein was localized in astrocytes and MMP-12 was identified in microglia, astrocytes, and cells of oligodendrocyte lineage. These findings suggest that MMPs and TIMPs have roles in the regulation of demyelination and remyelination in thismodel. Moreover, differences in the expression levels of these genesbetween white and gray matter reveal region-specific molecularmechanisms.

Cuprizone feeding is a commonly used model to study experimental de- and remyelination, with the corpus callosum being the most frequently investigated white matter tract. We have previously shown that demyelination is also extensive in the cerebral cortex in the cuprizone model. In the current study, we have performed a detailed analysis of the dynamics of demyelination in the cortex in comparison to the corpus callosum. Prominent and almost complete demyelination in the corpus callosum was observed after 4.5-5 weeks of 0.2% cuprizone feeding, whereas complete cortical demyelination was only observed after 6 weeks of cuprizone feeding. Interestingly, remyelination in the corpus callosum occurred even before the termination of cuprizone administration. Accumulation of microglia in the corpus callosum started as early as week 3 reaching its maximum at week 4.5 and was still significantly elevated at week 6 of cuprizone treatment. Within the cortex only a few scattered activated microglial cells were found. Furthermore, the intensity of astrogliosis, accumulation of oligodendrocyte progenitor cells and nestin positive cells differed between the two areas investigated. The time course and dynamics of demyelination differ in the corpus callosum and in the cortex, suggesting different underlying pathomechanisms.

Foxp3(+) regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss-of-function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue-specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M-CSF and other inflammatory cytokines, including monocyte-recruiting chemokines. Adoptive transfer of WT CD4(+) cells and in vivo neutralization of M-CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte-related cytokines in Sf mice, while Treg cell transfer to RAG2(-/-) mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.

Foxp3 + regulatory T cells (Tregs) play a pivotal role in establishing and maintaining immune tolerance under homeostatic and inflammatory conditions. A loss-of-function mutation in the Foxp3 gene results in Treg deficiency and causes Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome, a rare and life-threatening autoimmune disorder. Acute respiratory distress syndrome has been reported as a cause of death in several IPEX cases. For this reason, we investigated lungs in Scurfy (Sf) mice, a murine experimental model of IPEX. When compared to littermate-controls (Wt), Sf mice demonstrated high leukocyte influx to the lung with infiltrates located around larger airways, forming structures resembling bronchus-associated lymphoid tissue (BALT). This was accompanied with mucus secretion and increased Th1, Th2, and Th17 cytokine production by in vitro cultured lung cells. Sf mice had higher numbers of total lung cells and of CD3 + CD8 + lymphocytes. In comparison to the Wt controls, Sf mice displayed lower percentages of CD19 + B cells in the lung but higher B cell counts in the bronchoalveolar lavage. Furthermore, cytokine and chemokine levels were elevated in the bone marrow and serum of Sf mice. Adoptive transfer of Tregs to neonatal Sf mice significantly reduced pulmonary inflammation and mucus production and restored skewed frequencies of immune cells. Our findings illustrate the profound impact of Tregs on the lung response, which may help to understand the lung pathology in IPEX syndrome and, on the long run, to improve therapeutic approaches.

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor genes, resulting in disturbed alveolar macrophage differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. So far, the only effective treatment for herPAP is repetitive whole-lung lavage, a merely symptomatic and highly invasive procedure. We introduce pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for herPAP. In a murine disease model, intrapulmonary transplanted macrophage progenitors displayed selective, long-term pulmonary engraftment and differentiation into functional alveolar macrophages. A single transplantation ameliorated the herPAP phenotype for at least 9 months, resulting in significantly reduced alveolar proteinosis, normalized lung densities in chest computed tomography, and improved lung function. A significant and sustained disease resolution was also observed in a second, humanized herPAP model after intrapulmonary transplantation of human macrophage progenitors. The therapeutic effect was mediated by long-lived, lung-resident macrophages, which displayed functional and phenotypical characteristics of primary human alveolar macrophages. Our findings present the concept of organotopic transplantation of macrophage progenitors as an effective and long-lasting therapy of herPAP and may also serve as a proof of principle for other diseases, expanding current stem cell-based strategies toward potent concepts using the transplantation of differentiated cells.

Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental animal model commonly used to study de- and remyelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte death with subsequent demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown, and appropriate in vitro investigations to study these mechanisms are not available. Thus, we studied cuprizone effects on rat primary glial cell cultures and on the neuroblastoma cell line SH-SY5Y. Treatment of cells with different concentrations of cuprizone failed to show effects on the proliferation and survival of SH-SY5Y cells, microglia, astrocytes, and oligodendrocyte precursor cells (OPC). In contrast, differentiated mature oligodendrocytes (OL) were found to be significantly affected by cuprizone treatment. This was accompanied by a reduced mitochondrial potential in cuprizone-treated OL. These results demonstrate that the main toxic target for cuprizone is mature OL, whilst other glial cells including OPC are not or only marginally affected. This explains the selective demyelination induced by cuprizone in vivo.

The chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR2 have been found to be expressed on microglia in many neurodegenerative diseases, such as multiple sclerosis and Alzheimer's disease. There is emerging evidence that chemokines, besides chemoattraction, might directly modulate reactive profiles of microglia. To address this hypothesis we have investigated the effects of CCL2, CCL3, CCL5, and CXCL1 on cytokine and growth factor production, NO synthesis, and phagocytosis in non-stimulated and lipopolysaccharide-stimulated primary rat microglia. The respective receptors CCR1, CCR5, and CXCR2 were shown to be functionally expressed on microglia. All tested chemokines stimulated chemotaxis whereas only CCL5 increased NO secretion and attenuated IL-10 as well as IGF-1 production in activated microglia. Based on these findings we propose that besides its chemoattractant function CCL5 has a modulatory effect on activated microglia.

Glatiramer acetate (GA) is an approved immunomodulating agent for the treatment of relapsing-remitting multiple sclerosis. Its mode of action is attributed to a T helper cell-type 1 (Th1) to Th2 cytokine shift in T cells. Th2-type GA-reactive T cells migrate into the brain and act suppressive at the sites of inflammation. However, there is increasing evidence that the effect of GA is not confined to T cells. It inhibits broadly the activation of monocytes and induces peritoneal macrophages and monocytes to differentiate into a type 2 antigen-presenting cell (APC) secreting anti-inflammatory cytokines. Thus, we examined whether GA has also direct effects on microglia cells which are involved in modifying/directing the local microenvironment in the central nervous system. Primary rat microglia were purified and cultured under standard conditions. Griess reaction was used to measure one of the stable end products of nitric oxide (NO), nitrite. Tumor necrosis factor (TNF)-alpha and interleukin-10 (IL-10) were measured in the cell culture supernatants using ELISA. Phagocytosis was quantified with a FACS-based assay. Our experiments show that GA directly modulates microglia cells. It promotes the phagocytic activity and increases the secretion of IL-10 while it decreases that of TNFα. In contrast, there was no effect on NO production. GA induces a type 2 APC differentiation of microglia suggesting a general effect on myeloid monocytic cells. Using microglia we report for the first time that GA promotes phagocytosis which could play an important role in removal of debris.

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14(++)CD16(+) cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14(++)CD16(+) monocyte subset.

Demyelination is the cause of disability in various neurological disorders. It is therefore crucial to understand the molecular regulation of oligodendrocytes, the myelin forming cells in the CNS. Growth factors are known to be essential for the development and maintenance of oligodendrocytes and are involved in the regulation of glial responses in various pathological conditions. We employed the well established murine cuprizone model of toxic demyelination to analyze the expression of 13 growth factors in the CNS during de- and remyelination. The temporal mRNA expression profile during demyelination and the subsequent remyelination were analyzed separately in the corpus callosum and cerebral cortex using laser microdissection and real-time PCR techniques. During demyelination a similar pattern of growth factor mRNA expression was observed in both areas with a strong up-regulation of NRG1 and GDNF and a slight increase of CNTF in the first week of cuprizone treatment. HGF, FGF-2, LIF, IGF-I, and TGF-ß1 were up-regulated mainly during peak demyelination. In contrast, during remyelination there were regional differences in growth factor mRNA expression levels. GDNF, CNTF, HGF, FGF-2, and BDNF were elevated in the corpus callosum but not in the cortex, suggesting tissue differences in the molecular regulation of remyelination in the white and grey matter. To clarify the cellular source we isolated microglia from the cuprizone lesions. GDNF, IGF-1, and FGF mRNA were detected in the microglial fraction with a temporal pattern corresponding to that from whole tissue PCR. In addition, immunohistochemical analysis revealed IGF-1 protein expression also in the reactive astrocytes. CNTF was located in astrocytes. This study identified seven different temporal expression patterns for growth factors in white and grey matter and demonstrated the importance of early tissue priming and exact orchestration of different steps during callosal and cortical de- and remyelination.