Search

Objective To determine whether serum neurofilament light chain (sNfL) levels are suppressed in patients with the clinically isolated syndrome (CIS) following narrowband ultraviolet B phototherapy (UVB-PT). Methods sNfL levels were measured using a sensitive single-molecule array assay at baseline and up to 12 months in 17 patients with CIS, 10 of whom received UVB-PT, and were compared with healthy control (HC) and early relapsing remitting multiple sclerosis (RRMS) group. sNfL levels were correlated with magnetic resonance imaging total lesion volume (LV) determined using icobrain version 4.4.1 and with clinical outcomes. Results Baseline median sNfL levels were significantly higher in the CIS (20.6 pg/mL, interquartile range [IQR] 13.7–161.4) and RRMS groups (36.6 pg/ml [IQR] 16.2–212.2) than in HC (10.7 pg/ml [IQR] 4.9–21.5) (p = .012 and p = .0002, respectively), and were strongly correlated with T2 and T1 LV at 12 months (r = .800; p = .014 and r = .833; p = .008, respectively) in the CIS group. Analysis of changes in sNfL levels over time in the CIS group showed a significant cumulative suppressive effect of UVB-PT in the first 3 months (UVB-PT −10.6% vs non-UVB-PT +58.3%; p = .04) following which the levels in the two groups converged and continued to fall. Conclusions Our findings provide the basis for further studies to determine the utility of sNfL levels as a marker of neuro-axonal damage in CIS and early MS and for assessing the efficacy of new therapeutic interventions such as UVB-PT.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system that results in demyelination of axons, inefficient signal transmission and reduced muscular mobility. Recent findings suggest that B cells play a significant role in disease development and pathology. To further explore this, B cell profiles in peripheral blood from 28 treatment-naive patients with early MS were assessed using flow cytometry and compared to 17 healthy controls. Conventional and algorithm-based analysis revealed a significant increase in MS patients of IgA+ memory B cells (MBC) including CD27+, CD27- and Tbet+ subsets. Screening circulating B cells for markers associated with B cell function revealed a significantly decreased expression of the B cell activation factor receptor (BAFF-R) in MS patients compared to controls. In healthy controls, BAFF-R expression was inversely associated with abundance of differentiated MBC but this was not observed in MS. Instead in MS patients, decreased BAFF-R expression correlated with increased production of proinflammatory TNF following B cell stimulation. Finally, we demonstrated that reactivation of Epstein Barr Virus (EBV) in MS patients was associated with several phenotypic changes amongst MBCs, particularly increased expression of HLA-DR molecules and markers of a T-bet+ differentiation pathway in IgM+ MBCs. Together, these data suggest that the B cell compartment is dysregulated in MS regarding aberrant MBC homeostasis, driven by reduced BAFF-R expression and EBV reactivation. This study adds further insights into the contribution of B cells to the pathological mechanisms of MS, as well as the complex role of BAFF/BAFF-R signalling in MS.

B cells are critical to the development of multiple sclerosis (MS), but the mechanisms by which they contribute to the disease are poorly defined. We hypothesised that the expression of CD32b (FcγRIIb), a receptor for the Fc region of IgG with inhibitory activities in B cells, is lower on B cell subsets from people with clinically isolated syndrome (CIS) or MS. CD32b expression was highest on post-naive IgM+ B cell subsets in healthy controls. For females with MS or CIS, significantly lower CD32b expression was identified on IgM+ B cell subsets, including naive and IgMhi MZ-like B cells, when compared with control females. Lower CD32b expression on these B cell subsets was associated with detectable anti-Epstein Barr Virus viral capsid antigen IgM antibodies, and higher serum levels of B cell activating factor. To investigate the effects of lower CD32b expression, B cells were polyclonally activated in the presence of IgG immune complexes, with or without a CD32b blocking antibody, and the expression of TNF and IL-10 in B cell subsets was assessed. The reduction of TNF but not IL-10 expression in controls mediated by IgG immune complexes was reversed by CD32b blockade in naive and IgMhi MZ-like B cells only. However, no consequence of lower CD32b expression on these cells from females with CIS or MS was detected. Our findings highlight a potential role for naive and marginal zone-like B cells in the immunopathogenesis of MS in females, which requires further investigation.

Altered composition of gut bacteria and changes to the production of their bioactive metabolites, the short-chain fatty acids (SCFAs), have been implicated in the development of multiple sclerosis (MS). However, the immunomodulatory actions of SCFAs and intermediaries in their ability to influence MS pathogenesis are uncertain. In this study, levels of serum SCFAs were correlated with immune cell abundance and phenotype as well as with other relevant serum factors in blood samples taken at first presentation of Clinically Isolated Syndrome (CIS; an early form of MS) or MS and compared to healthy controls. There was a small but significant reduction in propionate levels in the serum of patients with CIS or MS compared with healthy controls. The frequencies of circulating T follicular regulatory cells and T follicular helper cells were significantly positively correlated with serum levels of propionate. Levels of butyrate associated positively with frequencies of IL-10-producing B-cells and negatively with frequencies of class-switched memory B-cells. TNF production by polyclonally-activated B-cells correlated negatively with acetate levels. Levels of serum SCFAs associated with changes in circulating immune cells and biomarkers implicated in the development of MS.

Objectives At the end of a 60‐day course of narrowband UVB phototherapy, administered to individuals with early multiple sclerosis, there were changes in the relative proportions of circulating B‐cell subsets. This study investigated phototherapy‐associated changes to cytokine responses of B cells when exposed to a TLR7 ligand. Methods PBMCs from participants of the PhoCIS (Phototherapy for Clinically Isolated Syndrome) trial taken before (day 1) and after phototherapy for 8 weeks (day 60) were incubated with, or without, the TLR7 ligand, R848, for 18 h. Production of TNF and IL‐10 in seven B‐cell subsets was examined, with cytokine responses in each individual at day 60, adjusted for responses at day 1. Paired PBMCs were from participants administered phototherapy (n = 7) or controls (n = 6). Results At day 60, significantly fewer B cells, particularly marginal zone‐like B cells (CD27+/IgD+), from participants administered phototherapy produced TNF in response to TLR7 stimulation. When responses by seven B‐cell subsets were analysed together using multivariate methods, a phototherapy‐specific signature was observed. An increased responsiveness from day 1 to day 60 in IgM‐only memory B cells (CD27+/IgD−/IgM+) after TLR7 stimulation also predicted slower progression from CIS to MS. Phototherapy was without significant effect on B‐cell IL‐10 production. Conclusions Reduced TNF responses after TLR7 stimulation in marginal zone‐like B cells from participants administered phototherapy suggested treatment‐associated priming effects that were detected upon subsequent polyclonal B‐cell activation. Changes in responsiveness to TLR7 stimulation also suggested that IgM‐only memory B cells may be important in conversion from CIS to MS.

Background: UV radiation exposure has immunosuppressive effects in humans. Low environmental UV radiation exposure is associated with multiple sclerosis risk. Narrowband UVB phototherapy is an established treatment for psoriasis, and is under investigation in people with clinically isolated syndrome (CIS) at risk of developing MS. Objective: To determine whether systemic leukocyte phenotypes in blood are significantly altered by a short course of narrowband UVB phototherapy in people with CIS. Methods: 20 participants with CIS were recruited and half received narrowband UVB phototherapy in 24 sessions over 2 months. Peripheral blood mononuclear cells (PBMC) were collected at baseline, and 1, 2, 3, 6 and 12 months after enrolment. Multicolour flow cytometry was used to identify T cell, B cell, monocyte, dendritic cell, and natural killer cell subsets. The effects of phototherapy on the frequencies of PBMCs were examined using linear regression. Results: Significant differences in the frequencies of PBMC phenotypes were detected between treated and untreated individuals during phototherapy, including in subsets of B cells, monocytes and T cells. No effects were detected after phototherapy treatment ceased. The largest treatment effects were detected in B cell subsets, where phototherapy was associated with significantly higher naïve B cell frequency and significantly lower frequency of switched memory B cells. Conclusions: Narrowband UVB phototherapy may contribute to immune suppression through regulation of systemic leukocyte phenotypes in people with CIS.

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.

Clinically isolated syndrome (CIS) is the earliest clinical episode in multiple sclerosis (MS). Low environmental exposure to UV radiation is implicated in risk of developing MS, and therefore, narrowband UVB phototherapy might delay progression to MS in people with CIS. Twenty individuals with CIS were recruited, and half were randomised to receive 24 sessions of narrowband UVB phototherapy over a period of 8 weeks. Here, the effects of narrowband UVB phototherapy on the frequencies of circulating immune cells and immunoglobulin levels after phototherapy are reported. Peripheral blood samples for all participants were collected at baseline, and 1, 2, 3, 6 and 12 months after enrolment. An extensive panel of leukocyte populations, including subsets of T cells, B cells, monocytes, dendritic cells, and natural killer cells were examined in phototherapy-treated and control participants, and immunoglobulin levels measured in serum. There were significant short-term increases in the frequency of naïve B cells, intermediate monocytes, and fraction III FoxP3+ T regulatory cells, and decreases in switched memory B cells and classical monocytes in phototherapy-treated individuals. Since B cells are increasingly targeted by MS therapies, the effects of narrowband UVB phototherapy in people with MS should be investigated further.

A proportion of circulating 25-hydroxy vitamin D3 (25(OH)D3)) undergoes epimerization to form C3-epi 25(OH)D3 and C3-epi 1,25(OH)2D3. These epimers have less calcaemic activity than non-epimerized metabolites and are not differentiated by many immunoassays when reporting total 25(OH)D3 levels. This study aimed to compare the effect of exposure to ultraviolet radiation (UVR) and oral vitamin D3 supplementation on vitamin D C3-epimer levels. C57Bl/6 female mice were fed either vitamin D-sufficient (vitamin D3 2000 IU/kg) or -deficient diets (no vitamin D3) for 4 weeks. Among the vitamin D-deficient group, the shaved backs of half were irradiated daily for 4 days with 1 kJ/m2 UVR, followed by twice weekly irradiation for 4 weeks. Despite similar 25(OH)D3 levels, the UV-irradiated group had a lower proportion of C3-epi 25(OH)D3 at week 7 (p < 0.05) and week 9 (p < 0.01). C3-epimer concentrations and %C3-epi 25(OH)D3 were also analysed in serum samples from two human clinical trials. These trials investigated the effect of high dose oral vitamin D3 supplementation and narrowband UVB phototherapy, respectively. Serum 25(OH)D3 and the %C3-epi 25(OH)D3 levels measured at 12 months after oral vitamin D3 supplementation were not significantly different to those measured at the time of maximal effect of phototherapy (2 months). Thus, the proportion of 25(OH)D3 that undergoes epimerization is greater with oral vitamin D3 supplementation than exposure to UVR in mice, but not in humans. This important difference between human and murine vitamin D metabolism warrants consideration when interpreting animal studies.

Clinically isolated syndrome (CIS) is a first episode of neurological symptoms that may precede a diagnosis of multiple sclerosis (MS). Therefore, studying individuals with CIS may lead to breakthroughs in understanding the development and pathogenesis of MS. In this study, serum levels of immunoglobulin (Ig)G, IgA, IgM, and IgG1–4 were measured in 20 people with CIS and compared with those in 10 healthy controls (HC) and 8 people with MS. Serum Ig levels in individuals with CIS were compared with (a) the time to their conversion from CIS to MS, (b) serum levels of antibodies to Epstein–Barr virus, (c) frequencies of T regulatory (Treg), T follicular regulatory (Tfr), and B cell subsets, and (d) Treg/Tfr expression of Helios. Serum IgG, IgM, and IgG2 levels were significantly lower in people with CIS than HC, and IgG, IgM, and IgG1 levels were significantly lower in people with CIS than MS. After adjusting for age, sex, and serum 25(OH) vitamin D3 [25(OH)D] levels, CIS was associated with lower serum levels of IgG and IgG2 compared with HC (p = 0.001 and p < 0.001, respectively). People with MS had lower IgG2 levels (p < 0.001) and IgG2 proportions (%IgG; p = 0.007) compared with HC. After adjusting for age, sex, and 25(OH)D, these outcomes remained, in addition to lower serum IgA levels (p = 0.01) and increased IgG3 levels (p = 0.053) in people with MS compared with HC. Furthermore, serum from people with MS had increased proportions of IgG1 and IgG3 (p = 0.03 and p = 0.02, respectively), decreased proportions of IgG2 (p = 0.007), and greater ratios of “upstream” to “downstream” IgG subclasses (p = 0.001) compared with HC. Serum IgG3 proportions (%IgG) from people with CIS correlated with the frequency of plasmablasts in peripheral blood (p = 0.02). Expression of Helios by Treg and Tfr cell subsets from individuals with CIS correlated with levels of serum IgG2 and IgG4. IgG3 levels and proportions of IgG3 (%IgG) in serum at CIS diagnosis were inversely correlated w

Objectives Clinically isolated syndrome (CIS) is the earliest clinical episode in multiple sclerosis (MS). A study of circulating cells from patients with CIS may help us understand the transition to, and processes associated with, the development of MS. Methods As immune cell activity can be determined by flux through metabolic pathways, the mRNA expression of l‐tryptophan‐ and l‐arginine‐catabolising enzymes, indoleamine 2,3‐dioxygenase (IDO) 1 and IDO2 and arginase (ARG) 1 and ARG2, respectively, was compared between peripheral blood mononuclear cells (PBMCs) from healthy controls, and patients with CIS and definite MS. As one measure of cell function, cytokine mRNA levels were analysed directly ex vivo and in cells after culture for 4 h in the absence of regulatory factors in autologous serum. Results When measured directly ex vivo, the expression of IDO and ARG was greater in cells from patients with CIS and MS than cells from healthy controls. Although not linked to IDO and ARG expression, PBMCs from the CIS patients were characterised by low IL‐10 and TGFB mRNA levels and not by greater expression of proinflammatory cytokines. When the cells were cultured for 4 h without autologous serum, pro‐ and anti‐inflammatory cytokine mRNA levels positively correlated with IDO1 expression, and TGFB mRNA levels correlated with ARG1 expression. Conclusion Higher IDO and ARG expression in CIS and MS provides one sustained homeostatic mechanism to control MS‐associated inflammation. However, potent extrinsic mediators in serum may regulate immune cell function in CIS and associations between IDO, ARG and cytokine expression.

Background The natural history of multiple sclerosis (MS) typically presents with the clinically isolated syndrome (CIS), an episode of neurological symptoms caused by central nervous system inflammation or demyelination that does not fulfil the diagnostic criteria for MS. Objective As preclinical studies have suggested that exposure to ultraviolet radiation (UVR) could regulate the development of MS, the Phototherapy for CIS (PhoCIS trial) was established to examine the effects of narrowband UVB phototherapy on patients with CIS, and their conversion to MS. Methods Of the 20 participants, half received 24 sessions of narrowband UVB exposure over eight weeks; participants in both arms were followed for 12 months. All participants were supplemented to 25-hydroxyvitamin D3 levels of >80 nmol/l. Results By 12 months, 100% of those in the no phototherapy arm and 70% in the phototherapy arm had converted to MS, although this difference was not statistically significant. Conclusion This study provides a basis for further studies to determine if there are any benefits of the therapeutic effects of narrowband UVB radiation on MS progression.

Background: Trials of vitamin D supplementation have to date lacked definitive outcomes in MS patients. Narrowband UVB can induce vitamin D production, but also other important immune-regulatory molecules in skin. Objective: The PhoCIS trial (Phototherapy for Clinically Isolated Syndrome) was established in Perth, Australia (32 degrees S), to investigate the clinical, radiological and immunological effects of narrowband UVB phototherapy on MS development in CIS. Patients and Methods: Nineteen individuals with CIS have been recruited with 53% of them given narrowband UVB phototherapy of 3 sessions per week for 8 weeks. All 19 participants were supplemented when necessary with vitamin D to 25(OH)-vitamin D levels of approximately 80 nmol/L. MRI was performed after 3, 6 and 12 months, and extensive blood cell phenotyping at 1 week, 1, 2, 3, 6 and 12 months after recruitment. No participant was taking any disease modifying drugs at recruitment. Results: After 6 months, 7 of 9 participants (78%) without phototherapy converted to MS (McDonald criteria). Only 5 of 10 participants (50%) who received phototherapy converted to MS (P=0.22). UVB therapy prevented the increase in memory B cells in the blood of non-phototherapy CIS participants, and produced a significant increase in immunoprotective IgG4. Conclusion: These interim results demonstrate UVB effects slowing the progression of individuals with CIS to MS. The PhoCIS trial provides a fresh approach to re-defining the reported associations of 25(OH)-vitamin D levels with MS development and progression. The outcomes suggest that UVB-irradiation of skin is immunomodulatory independent of Vitamin D, and can regulate CIS to MS progression.

Development of multiple sclerosis (MS) is frequently preceded by an acute or subacute neurological disturbance referred to as clinically isolated syndrome (CIS). The specific immunological disturbances present in CIS remain underexamined. This study analysed peripheral blood mononuclear cells from n=18 treatment-naive individuals with recently diagnosed CIS (<120 days) for disturbances in the phenotype of T regulatory (Treg), follicular T regulatory (Tfr), T helper (Th), follicular T helper (Tfh) and B cells. Relative to healthy controls (n=19), CIS was associated with lower proportions of suppressive CD45RA+FoxP3lo Treg and Tfr cells and greater proportions of non-suppressive CD45RA−FoxP3lo and Th17-like Treg and Tfr. Lower Helios expression (maen fluorescent intensity) was measured across all Treg and Tfr fractions in the CIS group, suggesting less potent regulatory function. Greater frequencies of activated, efficient B-cell helper Tfh subsets and a trend for a higher proportion of IgD−CD27− B cells was also detected in the CIS group, characteristics that were positively correlated with Treg and Tfr Helios expression. These results indicate that Treg and Tfr impairment is an early feature in MS.

Low vitamin D and insufficient sun exposure are additive independent risk factors for the development of multiple sclerosis (MS). The usual measure of vitamin D status, serum 25-hydroxy vitamin D [25(OH)D], is also a marker of recent exposure to the UVB rays of sunshine. The main evidence for a protective effect for MS development of higher 25(OH)D comes from observational studies, but this study design cannot separate out whether 25(OH)D is acting as a marker of vitamin D status, sun exposure, or both. In light of a lack of definitive outcomes in MS patients after trials of vitamin D supplementation and the ability of narrowband UVB to induce vitamin D, as well as other immune-regulatory molecules in skin, the Phototherapy for Clinically Isolated Syndrome (PhoCIS) trial was established to investigate the benefits of narrowband UVB, in addition to supplemented vitamin D, on MS development in individuals with Clinically Isolated Syndrome. We propose that the PhoCIS trial provides a fresh approach to re-defining the reported associations of 25(OH)D levels with MS development and progression.

Circulating T and B lymphocytes contribute to the pathogenesis of the neuroinflammatory autoimmune disease, multiple sclerosis (MS). Further progress in the development of MS treatments is dependent upon a greater understanding of the immunological disturbances that underlie the disease. Analyses of circulating immune cells by flow cytometry have revealed MS-associated alterations in the composition and function of T and B cell subsets, including temporal changes associated with disease activity. Disturbances in circulating immune populations reflect those observed in the central nervous system and include skewing towards proinflammatory CD4+ and CD8+ T cells and B cells, greater proportions of follicular T helper cells and functional defects in the corresponding T and B regulatory subsets. Utilizing the analytical power of modern flow cytometers, researchers are now well positioned to monitor immunological changes associated with disease activity or intervention, describe immunological signatures with predictive value and identify targets for therapeutic drug development. This review discusses the contribution of various T and B lymphocyte subsets to MS pathogenesis, provides current and relevant phenotypical descriptions to assist in experimental design and highlights areas of future research.

Background and Aims: Non-alcoholic fatty liver disease (NAFLD) and serum 25-hydroxyvitamin D (s25[OH]D) concentrations are both associated with adiposity and insulin resistance (IR) and thus may be pathogenically linked. We aimed to determine the prevalence of vitamin D deficiency in adolescents with NAFLD and to investigate the prospective and cross-sectional associations between s25[OH]D concentrations and NAFLD. Methods: Participants in the population-based West Australian Pregnancy (Raine) Cohort had seasonally adjusted s25(OH)D concentrations determined at ages 14 and then 17 years. NAFLD was diagnosed at 17 years using liver ultrasonography. Associations were examined after adjusting for potential confounders. Odds ratios (ORs) and confidence intervals (CIs) are reported per standard deviation in s25(OH)D concentrations. Results: NAFLD was present in 16% (156/994) of adolescents. The majority of participants with NAFLD had either insufficient (51%) or deficient (17%) vitamin D status. s25(OH)D concentrations at 17 years were inversely associated with risk of NAFLD (OR 0.74, 95% CI 0.56, 0.97; P=0.029), after adjusting for sex, race, physical activity, television/computer viewing, body mass index, and IR. The effect of s25(OH)D concentrations at 17 years was minimally affected after further adjusting for s25(OH)D concentrations at 14 years (OR 0.76, 95% CI 0.56, 1.03; P=0.072). Conclusions: Lower s25(OH)D concentrations are significantly associated with NAFLD, independent of adiposity and IR. Screening for vitamin D deficiency in adolescents at risk of NAFLD is appropriate, and clinical trials investigating the effect of vitamin D supplementation in the prevention and treatment of NAFLD may be warranted.

Vitamin D may be essential for restricting the development and severity of allergic diseases and asthma, but a direct causal link between vitamin D deficiency and asthma has yet to be established. We have developed a 'low dose' model of allergic airway disease induced by intraperitoneal injection with ovalbumin (1 μg) and aluminium hydroxide (0.2 mg) in which characteristics of atopic asthma are recapitulated, including airway hyperresponsiveness, antigen-specific immunoglobulin type-E and lung inflammation. We assessed the effects of vitamin D deficiency throughout life (from conception until adulthood) on the severity of ovalbumin-induced allergic airway disease in vitamin D-replete and -deficient BALB/c mice using this model. Vitamin D had protective effects such that deficiency significantly enhanced eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male but not female mice. Vitamin D also suppressed the proliferation and T helper cell type-2 cytokine-secreting capacity of airway-draining lymph node cells from both male and female mice. Supplementation of initially vitamin D-deficient mice with vitamin D for four weeks returned serum 25-hydroxyvitamin D to levels observed in initially vitamin D-replete mice, and also suppressed eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male mice. Using generic 16 S rRNA primers, increased bacterial levels were detected in the lungs of initially vitamin D-deficient male mice, which were also reduced by vitamin D supplementation. These results indicate that vitamin D controls granulocyte levels in the bronchoalveolar lavage fluid in an allergen-sensitive manner, and may contribute towards the severity of asthma in a gender-specific fashion through regulation of respiratory bacteria.

The anti-inflammatory actions of IL-4 in activated human monocytes may reflect transcriptional regulation of genes involved in TLR signaling pathways. Tailored gene arrays were conducted to profile the expression of 84 genes central to TLR-mediated signal transduction in human monocytes treated with the TLR4 ligand, LPS, with or without IL-4. In the first 3h, IL-4 down-regulated mRNA levels of LPS-induced inflammatory cytokines and chemokines, without altering mRNA levels of TLRs, TLR-related signaling molecules or multiple transcription factors. The down-regulation of inflammatory genes by IL-4 was preceded by an early up-regulation of IL-10 mRNA and protein and mRNA for receptor-interacting serine-threonine kinase 2 (RIPK2), the TLR homolog, RP105, and c-Maf, a transcription factor required for IL-10 gene expression. However, IL-4 still suppressed LPS-induced TNFα production in bone-marrow derived macrophages from IL10 -/- mice, and in the presence of a neutralizing antibody to IL-10 in human monocytes. The up-regulation of RIPK2 and RP105 mRNA by IL-4 occurred independently of IL-10. IL-4 maintained the ability to suppress LPS-induced TNFα and enhance IL-10 production in the presence of RIPK2 kinase inhibitors. Further, IL-4 failed to up-regulate expression of RP105 at the cell surface. In conclusion, the anti-inflammatory actions of IL-4 occur independently of IL-10, RP105, and the kinase activity of RIPK2.

Enhanced SOCS1 expression following exposure of murine macrophages to LPS implicated SOCS1 in the control of LPS-mediated signaling. SOCS1 regulates NFkB signaling in murine macrophages, blocking at the level of Mal or IkBa phosphorylation. We investigated the role of SOCS1 in regulating the production TNF by LPS and Pam3CSK4-activated primary human monocytes. Blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing SOCS1 (AdV-SOCS1), control vector (AdVGFP) or left untreated. AdV-SOCS1 monocytes were exposed to TLR4 and TLR2 ligands, LPS (500 ng/ml) or Pam3CSK4 (300 ng/ml). FACS analysis demonstrated infection efficiencies of 50_4% and 67_4% (n=16, mean _ SEM) of monocytes expressing AdV-GFP or AdVSOCS1 at MOI 50. AdV-SOCS1 blocked LPS and Pam3CSK4 induced TNF mRNA and protein production in a dose-dependent manner. In contrast, IL-6 and IL-10 production by AdV-SOCS1-infected monocytes was not blocked. AdV-SOCS1 also blocked LPS and Pam3CSK4- induced TNF production by macrophages isolated from synovial fluid. Infection efficiencies of 67_1% or 72_1% were obtained. Quantitative Western blot analysis revealed that the classically defined NFkB pathway was not altered at the level of IkBa or p65 activation. Furthermore, the kinetics of LPS and Pam3CSK4- induced IkBa phosphorylation and degradation in AdVSOCS1 monocytes remained unaffected (n=5 and 2 donors, respectively). Further, analysis of parallel MAPK pathways demonstrated no block in p38 or ERK MAPK pathways. These data suggest that SOCS1 regulation of LPS and Pam3CSK4-induced TNF production by human monocytes occurs downstream of TLRs, possibly at the level of transcription.

To the Editor...

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.