Mapping Autoantibodies in Children With Acute Rheumatic Fever.

Background: Acute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease. Methods: This study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand. Results: Global analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p < 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens. Conclusions: The broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

The Treatment of Acute Rheumatic Fever: Novel Use of Hydroxychloroquine.

Hydroxychloroquine (HCQ) suppresses an interleukin-1ß-granulocyte-macrophage colony-stimulating factor cytokine axis, reported to be dysregulated in peripheral blood mononuclear cells of acute rheumatic fever patients ex vivo. We describe HCQ treatment for 2 patients with rheumatic carditis and a protracted inflammatory course. HCQ was associated with control of inflammatory markers, control of pericarditis in first patient and stabilization of progressive carditis in the second patient.

An immunological perspective on rheumatic heart disease pathogenesis: more questions than answers.

Acute rheumatic fever (ARF) and the related rheumatic heart disease (RHD) are autoimmune diseases thought to be triggered by group A streptococcal (GAS) pharyngitis. RHD is a leading cause of mortality in the developing world. The strong epidemiological association between GAS throat infection and ARF is highly suggestive of causation, but does not exclude other infections as contributory. There is good evidence of both humoral and cellular autoreactivity and GAS/self cross-reactivity in established RHD. RHD pathogenesis could feasibly be triggered and driven by humoral and/or cellular molecular cross-reactivity between GAS and host cardiac tissues (molecular mimicry). However, good evidence of humoral pathogenicity is lacking and the specific triggering event for RHD remains unknown. It is likely that the critical immunological events leading to ARF/RHD occur at the point of contact between GAS and the immune system in the throat, strongly implicating the mucosal immune system in RHD pathogenesis. Additionally, there is circumstantial evidence that continued live GAS may play a role in ARF/RHD pathogenesis. We suggest that future avenues for study should include the exclusion of GAS components directly contributing to RHD pathogenesis; large genome-wide association studies of patients with RHD looking for candidate genes involved in RHD pathogenesis; genome-wide association studies of GAS from patients with ARF taken at diagnosis to look for characteristics of rheumatogenic strains; and performing case/control studies of GAS pharyngitis/ARF/patients with RHD, and controls to identify microbiological, immunological and environmental differences to elucidate RHD pathogenesis.

Post-infectious group A streptococcal autoimmune syndromes and the heart.

There is a pressing need to reduce the high global disease burden of rheumatic heart disease (RHD) and its harbinger, acute rheumatic fever (ARF). ARF is a classical example of an autoimmune syndrome and is of particular immunological interest because it follows a known antecedent infection with group A streptococcus (GAS). However, the poorly understood immunopathology of these post-infectious diseases means that, compared to much progress in other immune-mediated diseases, we still lack useful biomarkers, new therapies or an effective vaccine in ARF and RHD. Here, we summarise recent literature on the complex interaction between GAS and the human host that culminates in ARF and the subsequent development of RHD. We contrast ARF with other post-infectious streptococcal immune syndromes - post-streptococcal glomerulonephritis (PSGN) and the still controversial paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), in order to highlight the potential significance of variations in the host immune response to GAS. We discuss a model for the pathogenesis of ARF and RHD in terms of current immunological concepts and the potential for application of in depth "omics" technologies to these ancient scourges.

Synthesis and Toll-like receptor 4 (TLR4) activity of phosphatidylinositol dimannoside analogues.

A series of five PIM(2) analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM(2) with the corresponding ether or amide. An AcPIM(2) analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM(2)). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity. Removal of the inositol ring enhanced antagonist activity, and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced 2:2 TLR4/MD-2 signaling complex formation by PIM(2) represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.

Monosodium urate monohydrate crystal-recruited noninflammatory monocytes differentiate into M1-like proinflammatory macrophages in a peritoneal murine model of gout.

OBJECTIVE: To profile monosodium urate monohydrate (MSU) crystal-recruited monocyte inflammatory function during the course of in vivo differentiation, in a murine model of peritoneal MSU crystal-induced inflammation. METHODS: C57BL/6J mice were injected intraperitoneally with MSU crystals, and the peritoneal cells were harvested at different time points. The MSU crystal-recruited monocyte/macrophage population was analyzed for the expression of differentiation and activation markers, cytokine production following MSU crystal restimulation ex vivo and in vivo, expression of NLRP3-associated proteins (ASC, caspase 1) and pro-interleukin-1ß (proIL-1ß), and phagocytic capacity. RESULTS: Monocytes recruited 8 hours after MSU crystal stimulation (F4/80(low) Gr-1(int) 7/4+) exhibited poor phagocytic capacity, expressed low levels of proIL-1ß, and failed to produce proinflammatory cytokines in response to MSU crystal restimulation. In the absence of MSU crystal restimulation, differentiating monocytes produced low levels of transforming growth factor ß1 ex vivo, and this was abrogated following MSU crystal restimulation. Over time these cells developed a proinflammatory phenotype in vivo, characterized by the production of IL-1ß, tumor necrosis factor α, IL-6, CCL2 (monocyte chemotactic protein 1), and CXCL1 (cytokine-induced neutrophil chemoattractant) following ex vivo MSU crystal restimulation, and leading to IL-1ß production and cell infiltration following MSU crystal rechallenge in vivo. Proinflammatory function was associated with differentiation toward a macrophage phenotype (F4/80(high) Gr-1-7/4-), an increase in phagocytic capacity, and an increase in the expression of proIL-1ß. CONCLUSION: MSU crystal-recruited monocytes differentiate into proinflammatory M1-like macrophages in vivo. This proinflammatory macrophage phenotype is likely to play a key role in perpetuating inflammation in gouty arthritis in the presence of ongoing deposition of fresh MSU crystals.

Chemical synthesis and immunosuppressive activity of dipalmitoyl phosphatidylinositol hexamannoside.

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannoside (PIM(6)) 1 and the first reported functional biology of a synthetic PIM(6). Key steps in the synthetic protocol included the selective glycosylation of an inositol 2,6-diol with a suitably protected mannosyl donor and construction of the glycan core utilizing a [3 + 4] thio-glycosylation strategy. The target 1 was purified by reverse phase chromatography and characterized by standard spectroscopic methods, HPLC, and chemical modification by deacylation to dPIM(6). The (1)H NMR spectrum of synthetic dPIM(6) obtained from 1 matched that of dPIM(6) obtained from nature. PIM(6) (1) exhibited dendritic cell-dependent suppression of CD8(+) T cell expansion in a human mixed lymphocyte reaction consistent with the well established immunosuppressive activity of whole mycobacteria.

Differences in MSU-induced superoxide responses by neutrophils from gout subjects compared to healthy controls and a role for environmental inflammatory cytokines and hyperuricemia in neutrophil function and survival.

OBJECTIVE: To determine whether monosodium urate (MSU) crystal-induced superoxide production is greater for neutrophils from patients with gout compared to asymptomatic hyperuricemic and healthy controls, and whether neutrophil functions are altered by an MSU crystal-induced inflammatory environment. METHODS: Neutrophils were purified from the whole blood of study participants, restimulated with 500 mg MSU crystals ex vivo, and superoxide production measured using the colorimetric dye WST-1. Purified neutrophils were exposed to conditioned media from MSU crystal-activated blood monocyte cultures with and without neutralizing antibodies for interleukin 1ss (IL-1ss), IL-8 (CXCL8), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Neutrophil superoxide production was measured and neutrophil apoptosis and IL-8 production were determined by flow cytometry. Serum samples were collected from participants and analyzed by Lincoplex bead array for IL-1ss, IL-8, IL-6, and TNF-alpha. RESULTS: Neutrophils from gout and asymptomatic hyperuricemic subjects produced higher levels of MSU crystal-induced superoxide, and a weak trend toward elevated serum cytokines was observed compared to healthy controls. A correlation between serum uric acid and elevated neutrophil superoxide production was also observed. Neutrophils exposed to media from MSU crystal-activated monocytes exhibited enhanced superoxide production to MSU-crystal stimulation, increased IL-8 production, and extended cell survival that was predominantly dependent on IL-8, TNF-alpha and IL-6, respectively. CONCLUSION: Neutrophils from gout and asymptomatic hyperuricemic individuals are primed for enhanced MSU crystal-induced superoxide production that may be driven by a subclinical inflammatory cytokine environment combined with hyperuricemia. This inflammatory environment likely contributes to elevated neutrophil IL-8 production and survival in the absence of direct crystal stimulation. Asymptomatic hyperuricemia is not associated with suppressed neutrophil function.

The anti-cancer, anti-inflammatory and tuberculostatic activities of a series of 6,7-substituted-5,8-quinolinequinones.

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discovery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone (10a) and 6-amino-7-methylsulfonyl-5,8-quinolinequinone (12), that exhibited selectivity for leukemic cells over T-cells, a highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone (18a) exhibiting the highest AI activity (0.11 microM), while 6,7-dichloro-5,8-quinolinequinone (7a), 6,7-dichloro-2-methyl-5,8-quinolinequinone (7b), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol (19) also exhibited good AI activity and specificity. Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolinequinone (11) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone (14), exhibited low MICs (1.56-3.13 microg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the functional group substitution of the quinolinequinone core and biological activity were also identified.

Innate inflammation and resolution in acute gout.

Acute gout is an inflammatory arthritis that is controlled by the innate arm of the immune response. Although the causative feature of gout has long been recognized, it is surprising that the cellular activities that underpin the initiation and resolution of acute gout remain poorly described. This review article summarizes what are currently thought to be the key cellular mechanisms at play during an inflammatory episode of acute gout. The emerging role of mononuclear phagocytes is highlighted as having a central role in both the initiation and resolution of acute gout, and the interplay between monocytes and other elements of the innate immune response, including neutrophils, and complement protein activation are discussed.

Sesquiterpene dialdehydes inhibit MSU crystal-induced superoxide production by infiltrating neutrophils in an in vivo model of gouty inflammation.

A hallmark feature of monosodium urate (MSU) crystal-induced inflammation in gouty arthritis is the infiltration of activated neutrophils into the joint. Therefore inhibition of neutrophil superoxide production is a rational target for treating inflammation in gout. The natural product polygodial and related sesquiterpene dialdehyde analogs were tested in vitro and in vivo for their ability to inhibit neutrophil infiltration and superoxide production in response to MSU crystal stimulation. Polygodial and other sesquiterpene dialdehydes exhibited dose-dependent inhibition of MSU-induced superoxide production in the micromolar and submicromolar ranges. Inhibition of superoxide production was dependent on the presence of the dialdehyde functional groups and was sensitive to blockade with the thiol-containing amino acid cysteine. Polygodial, 6-hydroxypaxidal and sesquiterpene 2 inhibited both neutrophil infiltration and neutrophil superoxide production in an MSU crystal-induced mouse model of gouty inflammation. Together, these data highlight the potential of sesquiterpene dialdehydes for development as anti-inflammatory agents for the treatment of neutrophil-driven inflammatory diseases including gout.

Resident macrophages initiating and driving inflammation in a monosodium urate monohydrate crystal-induced murine peritoneal model of acute gout.

OBJECTIVE: To determine whether infiltrating monocytes, neutrophils, or resident macrophages contribute to the early inflammatory response to monosodium urate monohydrate (MSU) crystals in vivo. METHODS: MSU crystal-induced inflammation was monitored using a peritoneal model of acute gout. The production of proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha [TNFalpha], IL-6) by resident macrophages, infiltrating monocytes, and neutrophils during the onset of gout was determined by flow cytometry. Infiltrating and resident peritoneal cells were cultured with MSU crystals ex vivo, and proinflammatory cytokine production was determined by multiplex cytokine array. Activated macrophages on the visceral epithelial lining of the peritoneum were identified by immunofluorescence histochemistry. The inflammatory immune response to MSU crystals was then compared with the inflammatory response in mice depleted of resident macrophages by pretreatment with clodronate liposomes. RESULTS: The production of cytokines in vivo preceded the influx of Gr-1(intermediate)7/4+ monocytes. Monocytes and neutrophils recruited during the inflammatory phase of the response to MSU crystals failed to produce proinflammatory cytokines either in vivo, or ex vivo following restimulation with MSU crystals. Stimulation of the naive peritoneal resident cell population with MSU crystals ex vivo resulted in positive staining of resident macrophages for the proinflammatory cytokines IL-1beta, TNFalpha, and IL-6. Depletion of the resident macrophage population resulted in a significant decrease in both MSU crystal-induced neutrophil infiltration and proinflammatory cytokine production in vivo despite the presence of infiltrating monocytes. CONCLUSION: These data indicate that resident macrophages, rather than infiltrating monocytes or neutrophils, are important for initiating and driving the early proinflammatory phase of acute gout.