Petar Lenert

CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-κB activation

Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.

Identification of methylated CpG motifs as inhibitors of the immune stimulatory CpG motifs

The unmethylated CpG motifs within E. coli DNA (EC) cause immune stimulation. In contrast, mammalian DNA such as calf thymus (CT) DNA had been thought to be immunologically inert. In this article, we demonstrate that CT DNA unexpectedly specifically inhibits the immune activation by EC but not that by endotoxin. This inhibitory effect was mediated in the signaling pathway activated by EC since CT DNA markedly inhibited the CpG-induced nuclear translocation of the transcription factors, NF-κB and AP-1. In addition, CT DNA significantly inhibited the synergistic immune activation by EC and endotoxin. The mechanism of the inhibition by CT DNA probably did not involve the inhibition of the cellular uptake of EC. Using a CpG-depleted plasmid, we demonstrated that CpG methylation played an important role in the inhibition by CT DNA. Compared with unmethylated plasmid DNA, CpG-methylated DNA inhibited the immune activation by EC to the same extent as did CT DNA. Importantly, the inhibitory effect of CT DNA was also observed in vivo. Our results suggest that methylated DNA may be applied to alleviate the unwanted immune stimulation and inflammation in systemic inflammatory response syndrome and in gene therapy with plasmid DNA.

Structural characterization of the inhibitory DNA motif for the type A (D)-CpG-induced cytokine secretion and NK-cell lytic activity in mouse spleen cells

Oligodeoxynucleotides (ODN) with the CpG motif have been shown to be potent stimulators of innate immunity. A theoretical concern is that uncontrolled stimulation of the innate immune system through the TLR-9 receptor could induce, or worsen, some autoimmune diseases such as adjuvant arthritis or systemic lupus erythematosus. Safe therapeutic use of such ODN could be enhanced if one could regulate some of their stimulatory activities. We have designed a group of synthetic ODNs, which were able to inhibit the induction of NK lytic activity, IL-12p40 and IFN-gamma cytokine secretion by type A (D)-CpG-ODNs. Inhibition occurred in both DNA-sequence and dose-dependent fashion. Fifty percent inhibition was achieved with ~10-nM concentration of the most potent inhibitory ODNs. Delayed addition of these ODNs for up to 2 h was still able to profoundly affect CpG-induced IL-12p40 production at 18 h. Inhibitory DNA motif consists of two nucleotide triplets, a proximal pyrimidine-rich CCT sequence and a more distal GGG triplet. Optimal distance between these blocks is between three to five nucleotides. The linker sequence between the CCT and GGG blocks can additionally modify the activity of inhibitory ODNs, in both a positive and in negative way. When the order of CCT and GGG blocks is reversed, inhibition is completely lost. These findings suggest that CpG regulation of innate immunity can itself be regulated by particular motifs, which could be of therapeutic benefit in autoimmune diseases.

FcγRIIB regulation of BCR/TLR-dependent autoreactive B-cell responses

Crosslinking of Fc γ receptor II B (FcγRIIB) and the BCR by immune complexes (IC) can downregulate antigen‐specific B‐cell responses. Accordingly, FcγRIIB deficiencies have been associated with B‐cell hyperactivity in patients with systemic lupus erythematosus and mouse models of lupus. However, we have previously shown that murine IgG2a‐autoreactive AM14 B cells respond robustly to chromatin‐associated IC through a mechanism dependent on both the BCR and the endosomal TLR9, despite FcγRIIB coexpression. To further evaluate the potential contribution of FcγRIIB to the regulation of autoreactive B cells, we have now compared the IC‐triggered responses of FcγRIIB‐deficient and FcγRIIB‐sufficient AM14 B cells. We find that FcγRIIB‐deficient cells respond significantly better than FcγRIIB‐sufficient cells when stimulated with DNA IC that incorporate low‐affinity TLR9 ligand (CG‐poor dsDNA fragments). AM14 B cells also respond to RNA‐associated IC through BCR/TLR7 coengagement, but such BCR/TLR7‐dependent responses are normally highly dependent on IFN‐α costimulation. However, we now show that AM14 FcγRIIB−/− B cells are very effectively activated by RNA IC without supplemental IFN‐α priming. These results demonstrate that FcγRIIB can effectively modulate both BCR/TLR9 and BCR/TLR7 endosomal‐dependent activation of autoreactive B cells.

Higher‐order CpG‐DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single‐stranded CpG‐oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG‐ODN capable of forming higher‐order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell‐activating factor, or simultaneous co‐engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF‐1, IL‐6 and IL‐12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self‐derived hypomethylated double‐stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co‐engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9‐induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

B cells do not take up bacterial DNA: an essential role for antigen in exposure of DNA to toll-like receptor-9

Murine dendritic cells (DC) and macrophages respond to bacterial CpG DNA through toll‐like receptor 9 (TLR9). Although it is frequently assumed that bacterial DNA is a direct stimulus for B cells, published work does not reliably show responses of purified B cells. Here we show that purified splenic B cells did not respond to Escherichia coli DNA with induction of CD86, despite readily responding to single‐stranded (ss) phosphodiester CpG oligodeoxynucleotides (ODN). This was due to a combination of weak responses to both long and double‐stranded (ds) DNA. B‐cell DNA uptake was greatly reduced with increasing DNA length. This contrasts with macrophages where DNA uptake and subsequent responses were enhanced with increasing DNA length. However, when DNA was physically linked to hen egg lysozyme (HEL), HEL‐specific B cells showed efficient uptake of DNA, and limited proliferation in response to the HEL–DNA complex. We propose that, in the absence of other signals, B cells have poor uptake and responses to long dsDNA to prevent polyclonal activation. Conversely, when DNA is physically linked to a B‐cell receptor (BCR) ligand, its uptake is increased, allowing TLR9‐dependent B‐cell activation in an antigen‐specific manner. We could not generate fragments of E. coli DNA by limited DNaseI digestion that could mimic the stimulatory effect of ss CpG ODN on naïve B cells. We suggest that the frequently studied polyclonal B‐cell responses to CpG ODN are relevant to therapeutic applications of phosphorothioate‐modified CpG‐containing ODN, but not to natural responses to foreign or host dsDNA.

Modulating toll-like receptor 7 and 9 responses as therapy for allergy and autoimmunity.

Type I allergic diseases, such as allergic rhinitis and asthma, depend on allergen-induced T-helper type 2 (Th2) cells and IgE-secreting plasma cells. Fortunately, this harmful immune response can be modified by engaging Toll-like receptor (TLR)7 and TLR9, offering hopes to allergy sufferers. While clinical trials employing synthetic ligands for TLR7 or TLR9 are under way, one can wonder whether TLR7 or TLR9 engagements may trigger inadvertent autoreactivity and/or Th1-/Th17-mediated tissue pathology. To neutralize such danger, we have pioneered the development of potent TLR9 pathway antagonists, inhibitory oligonucleotides (INH-ODNs), which work in a sequence-specific manner. Interestingly, INH-ODNs also have TLR7-inhibitory properties; however, these effects appear to be sequence independent and phosphorothioate backbone dependent. In B cells, co-engagement of the B-cell receptor for antigen and TLR7 or TLR9 may influence how INH-ODNs impose their regulatory effects. INH-ODNs block TLR9 activation by competitively antagonizing ligand binding to proteolytically cleaved C-terminal TLR9 fragment. One may envision future use of INH-ODNs in systemic autoimmune diseases, DNA-mediated sepsis, or other situations in which chronic inflammation results from abnormal TLR7- and/or TLR9-mediated immune activation.

Spotlight on blisibimod and its potential in the treatment of systemic lupus erythematosus: evidence to date

B cells in general and BAFF (B cell activating factor of the tumor necrosis factor [TNF] family) in particular have been primary targets of recent clinical trials in systemic lupus erythematosus (SLE). In 2011, belimumab, a monoclonal antibody against BAFF, became the first biologic agent approved for the treatment of SLE. Follow-up studies have shown excellent long-term safety and tolerability of belimumab. In this review, we critically analyze blisibimod, a novel BAFF-neutralizing agent. In contrast to belimumab that only blocks soluble BAFF trimer but not soluble 60-mer or membrane BAFF, blisibimod blocks with high affinity all three forms of BAFF. Furthermore, blisibimod has a unique structure built on four high-affinity BAFF-binding peptides fused to the IgG1-Fc carrier. It was tested in phase I and II trials in SLE where it showed safety and tolerability. While it failed to reach the primary endpoint in a recent phase II trial, post hoc analysis demonstrated its efficacy in SLE patients with higher disease activity. Based on these results, blisibimod is currently undergoing phase III trials targeting this responder subpopulation of SLE patients. The advantage of blisibimod, compared to its competitors, lies in its higher avidity for BAFF, but a possible drawback may come from its immunogenic potential and the anticipated loss of efficacy over time.

Tapering biologics in rheumatoid arthritis: a pragmatic approach for clinical practice

Optimal rheumatoid arthritis (RA) therapy in daily clinical practice is based on the treat-to-target strategy. Quicker escalation of therapy and earlier introduction of biological disease-modifying anti-rheumatic drugs have led to improved outcomes in RA. However, chronic immunosuppressive therapy is associated with adverse events and higher costs. In addition, our patients frequently express a desire for lower dosing and drug holidays. Current clinical practice guidelines from the American College of Rheumatology and European League Against Rheumatism suggest that rheumatologists consider tapering treatment after achieving remission. However, the optimal approach for tapering therapy in RA, specifically de-escalation of biologics, remains unknown. This clinical review discusses biologic tapering strategies in RA. We draw our recommendations for everyday clinical practice from the most recent observational, pragmatic, and controlled clinical trials on de-escalation of biologics in RA. For each biologic, we highlight clinically relevant outcomes, such as flare rates, recapture of the disease control with retreatment, radiographic progression, side effects, and functional impact. We discuss the use of musculoskeletal ultrasound to select patients for successful tapering. In conclusion, we provide the reader with a practical guide for tapering biologics in the rheumatology clinic.

Joint Hypermobility Syndrome: Recognizing a Commonly Overlooked Cause of Chronic Pain

Joint hypermobility syndrome, also known as benign hypermobility syndrome, is a connective tissue disease characterized by joint instability, chronic pain, and minor skin changes. It shares many clinical features of Ehlers-Danlos syndrome, Hypermobility Type; enough so that many authorities consider them as one disease process. Approximately 3% of the general population is believed to have joint hypermobility syndrome, but despite this high prevalence, due to lack of awareness, heterogeneity of clinical presentation, and reliance on physical examination for diagnosis, it is largely overlooked by primary care physicians as well as by specialists. This leads to delayed or missed opportunities for diagnosis, and inappropriate interventions that frustrate both providers and patients. We review the literature regarding the pathophysiology, diagnosis, treatment options, and prognosis of joint hypermobility syndrome, and advocate for primary care physicians to consider it in the differential diagnosis of patients with chronic pain.