Hendrik J. Nel

Association between tuberculosis and a polymorphic NFκB binding site in the interferon γ gene

Summary Interferon γ is believed to be crucial for host defence against many infections. To test the hypothesis that a polymorphism in the gene for interferon γ ( IFNG ) is associated with susceptibility to tuberculosis, we did two independent investigations. In a case-control study of 313 tuberculosis cases, we noted a significant association between a polymorphism (+874A→T) in IFNG and tuberculosis in a South African population (p=0·0055). This finding was replicated in a family-based study, in which the transmission disequilibrium test was used in 131 families (p=0·005). The transcription factor NFκB binds preferentially to the +874T allele, which is over-represented in controls. This preferential binding suggests that genetically determined variability in interferon γ and expression might be important for the development of tuberculosis.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis

A comprehensive structural portrait of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in rheumatoid arthritis.

Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients.

Citrullinated peptide-exposed DCs induced immune regulatory effects in HLA risk genotype–positive RA patients. Immunotherapy out of joint Autoantibodies to anti–citrullinated peptides (ACPA) are found in most patients with rheumatoid arthritis (RA), especially those with HLA-DRB1 risk alleles. Benham et al. report a first-in-human phase 1 trial of a single injection of autologous dendritic cells modified with an NF-κB inhibitor that have been exposed to four citrullinated peptide antigens. They find that HLA risk genotype–positive RA patients had reduced numbers of effector T cells and decreased production of proinflammatory cytokines compared with untreated RA patient controls. The therapy was safe and did not induce disease flares. These data support larger studies of antigen-specific immunotherapy for RA. In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritis in an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti–citrullinated peptide autoantibodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated with HLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCs modified with a nuclear factor κB (NF-κB) inhibitor exposed to four citrullinated peptide antigens, designated “Rheumavax,” in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered once intradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype–positive RA patients with citrullinated peptide–specific autoimmunity. Sixteen RA patients served as controls. Rheumavax was well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a reduction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15 (IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin447–455–Cit450 relative to controls. Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreased within 1 month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety and biological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides, and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen immunomodulatory therapy in RA.

T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles

IntroductionAnti-citrullinated peptide antibodies are found in rheumatoid arthritis (RA) patients with HLA-DRβ chains encoding the shared epitope (SE) sequence. Citrullination increases self-antigen immunogenicity, through increased binding affinity to SE-containing HLA-DR molecules. To characterise T-cell autoreactivity towards citrullinated self-epitopes, we profiled responses of SE+ healthy controls and RA patients to citrullinated and unmodified epitopes of four autoantigens.MethodsWe compared T-cell proliferative and cytokine responses to citrullinated and native type II collagen 1,237 to 1,249, vimentin 66 to 78, aggrecan 84 to 103 and fibrinogen 79 to 91 in six SE+ healthy controls and in 21 RA patients with varying disease duration. Cytokine-producing cells were stained after incubation with peptide in the presence of Brefeldin-A.ResultsAlthough proliferative responses were low, IL-6, IL-17 and TNF were secreted by CD4+ T cells of SE+ RA patients and healthy controls, as well as IFNγ and IL-10 secreted by RA patients, in response to citrullinated peptides. Of the epitopes tested, citrullinated aggrecan was most immunogenic. Patients with early RA were more likely to produce IL-6 in response to no epitope or to citrullinated aggrecan, while patients with longstanding RA were more likely to produce IL-6 to more than one epitope. Cytokine-producing CD4+ T cells included the CD45RO+ and CD45RO- and the CD28+ and CD28- subsets in RA patients.ConclusionProinflammatory cytokines were produced by CD4+ T cells in SE+ individuals in response to citrullinated self-epitopes, of which citrullinated aggrecan was most immunogenic. Our data suggest that the T-cell response to citrullinated self-epitopes matures and diversifies with development of RA.

SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses

SOCS2-deficient T cells more readily produce Th2 cytokines, and SOCS2-deficient mice exhibit exacerbated atopic dermatitis and allergic airway inflammation.

Identification of the Synthetic Cannabinoid R(+)WIN55,212-2 as a Novel Regulator of IFN Regulatory Factor 3 Activation and IFN-β Expression RELEVANCE TO THERAPEUTIC EFFECTS IN MODELS OF MULTIPLE SCLEROSIS

β-Interferons (IFN-βs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.β-Interferons (IFN-βs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.

Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens

BackgroundThe global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection.ResultsWe demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice.ConclusionOur data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production.

Dexamethasone and monophosphoryl lipid a induce a distinctive profile on monocyte-derived dendritic cells through transcriptional modulation of genes associated with essential processes of the immune response

There is growing interest in the use of tolerogenic dendritic cells (tolDCs) as a potential target for immunotherapy. However, the molecular bases that drive the differentiation of monocyte-derived DCs (moDCs) toward a tolerogenic state are still poorly understood. Here, we studied the transcriptional profile of moDCs from healthy subjects, modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), referred to as Dex-modulated and MPLA-activated DCs (DM-DCs), as an approach to identify molecular regulators and pathways associated with the induction of tolerogenic properties in tolDCs. We found that DM-DCs exhibit a distinctive transcriptional profile compared to untreated (DCs) and MPLA-matured DCs. Differentially expressed genes downregulated by DM included MMP12, CD1c, IL-1B, and FCER1A involved in DC maturation/inflammation and genes upregulated by DM included JAG1, MERTK, IL-10, and IDO1 involved in tolerance. Genes related to chemotactic responses, cell-to-cell signaling and interaction, fatty acid oxidation, metal homeostasis, and free radical scavenging were strongly enriched, predicting the activation of alternative metabolic processes than those driven by counterpart DCs. Furthermore, we identified a set of genes that were regulated exclusively by the combined action of Dex and MPLA, which are mainly involved in the control of zinc homeostasis and reactive oxygen species production. These data further support the important role of metabolic processes on the control of the DC-driven regulatory immune response. Thus, Dex and MPLA treatments modify gene expression in moDCs by inducing a particular transcriptional profile characterized by the activation of tolerance-associated genes and suppression of the expression of inflammatory genes, conferring the potential to exert regulatory functions and immune response modulation.

Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

Tolerogenic dendritic cells (TolDCs) are promising tools for therapy of autoimmune diseases, such as rheumatoid arthritis (RA). Here, we characterize monocyte-derived TolDCs from RA patients modulated with dexamethasone and activated with monophosphoryl lipid A (MPLA), referred to as MPLA-tDCs, in terms of gene expression, phenotype, cytokine profile, migratory properties, and T cell-stimulatory capacity in order to explore their suitability for cellular therapy. MPLA-tDCs derived from RA patients displayed an anti-inflammatory profile with reduced expression of co-stimulatory molecules and high IL-10/IL-12 ratio, but were capable of migrating toward the lymphoid chemokines CXCL12 and CCL19. These MPLA-tDCs induced hyporesponsiveness of autologous CD4+ T cells specific for synovial antigens in vitro. Global transcriptome analysis confirmed a unique transcriptional profile of MPLA-tDCs and revealed that RA-associated genes, which were upregulated in untreated DCs from RA patients, returned to expression levels of healthy donor-derived DCs after treatment with dexamethasone and MPLA. Thus, monocyte-derived DCs from RA patients have the capacity to develop tolerogenic features at transcriptional as well as at translational level, when modulated with dexamethasone and MPLA, overcoming disease-related effects. Furthermore, the ability of MPLA-tDCs to impair T cell responses to synovial antigens validates their potential as cellular treatment for RA.

Airway cells from protracted bacterial bronchitis and bronchiectasis share similar gene expression profiles

Protracted bacterial bronchitis (PBB) is a common cause of prolonged cough in young children, and may be a precursor of bronchiectasis. Bacteria are often present in the lower airways in both PBB and bronchiectasis and may cause persistent infections. However, there is a paucity of information available on the pathogenesis of PBB and the factors associated with persistent bacterial infection and progression to bronchiectasis. This study hypothesised that lung immune cells in recurrent PBB and bronchiectasis differentially express genes related to immune cell dysfunction compared to lung immune cells from control subjects.