Cardiac myosin-Th17 responses promote heart failure in human myocarditis.

In human myocarditis and its sequela dilated cardiomyopathy (DCM), the mechanisms and immune phenotype governing disease and subsequent heart failure are not known. Here, we identified a Th17 cell immunophenotype of human myocarditis/DCM with elevated CD4+IL17+ T cells and Th17-promoting cytokines IL-6, TGF-ß, and IL-23 as well as GM-CSF-secreting CD4+ T cells. The Th17 phenotype was linked with the effects of cardiac myosin on CD14+ monocytes, TLR2, and heart failure. Persistent heart failure was associated with high percentages of IL-17-producing T cells and IL-17-promoting cytokines, and the myocarditis/DCM phenotype included significantly low percentages of FOXP3+ Tregs, which may contribute to disease severity. We demonstrate a potentially novel mechanism in human myocarditis/DCM in which TLR2 peptide ligands from human cardiac myosin stimulated exaggerated Th17-related cytokines including TGF-ß, IL-6, and IL-23 from myocarditic CD14+ monocytes in vitro, and an anti-TLR2 antibody abrogated the cytokine response. Our translational study explains how an immune phenotype may be initiated by cardiac myosin TLR ligand stimulation of monocytes to generate Th17-promoting cytokines and development of pathogenic Th17 cells in human myocarditis and heart failure, and provides a rationale for targeting IL-17A as a therapeutic option.

Antineuronal antibodies in a heterogeneous group of youth and young adults with tics and obsessive-compulsive disorder.

BACKGROUND AND OBJECTIVE: Antineuronal antibodies have been implicated in tic and obsessive compulsive disorders (OCD) associated with group A streptococcal infections. We investigated antineuronal autoantibody levels as well as antibody-mediated neuronal cell signaling activity, as previously reported for Sydenham chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococci (PANDAS), to determine immunological profiles for a large cohort of children with tics and/or OCD. METHODS: Study participants (n=311; ages 4-27 years, 66% male) were selected from a larger group of individuals with self-reported neuropsychiatric symptoms (n=742) and included only those with accurate knowledge of group A streptococcal infection status, except for four individuals in whom streptococcal infection status was unknown. Healthy control samples (n=16; ages 5-14 years, 81% male), came from the National Institute of Mental Health and Yale University. In addition to serum donations, participants and/or legal guardians provided neuropsychiatric and related medical histories of symptoms that had lasted >1 year. Antineuronal immunoglobulin G (IgG) titers were measured by standard enzyme-linked immunosorbent assay (ELISA) and compared with mean titers of normal age-matched sera against lysoganglioside, tubulin, and dopamine receptors (D1R and D2R). Antibody-mediated signaling of calcium calmodulin dependent protein kinase II (CaMKII) activity in a human neuronal cell line (SK-N-SH) was tested in serum. RESULTS: Of 311 individuals, 222 (71%) had evidence of group A streptococcal infection, which was associated with tics and/or OCD status (p=0.0087). Sera from individuals with tics and/or OCD (n=261) had evidence of elevated serum IgG antibodies against human D1R (p<0.0001) and lysoganglioside (p=0.0001), and higher serum activation of CaMKII activity (p<0.0001) in a human neuronal cell line compared with healthy controls (n=16). Furthermore, patients with tics and OCD had significantly increased activation of CaMKII activity compared with patients with only tics or only OCD (p<0.033 for each). CONCLUSION: Our study suggested a significant correlation of streptococcal-associated tics and OCD with elevated anti-D1R and antilysoganglioside antineuronal antibodies in serum concomitant with higher activation of CaMKII in human neuronal cells. Youth and young adults with chronic tics and OCD may have underlying infectious/immunologic etiology.

Brain human monoclonal autoantibody from sydenham chorea targets dopaminergic neurons in transgenic mice and signals dopamine D2 receptor: implications in human disease.

How autoantibodies target the brain and lead to disease in disorders such as Sydenham chorea (SC) is not known. SC is characterized by autoantibodies against the brain and is the main neurologic manifestation of streptococcal-induced rheumatic fever. Previously, our novel SC-derived mAb 24.3.1 was found to recognize streptococcal and brain Ags. To investigate in vivo targets of human mAb 24.3.1, VH/VL genes were expressed in B cells of transgenic (Tg) mice as functional chimeric human VH 24.3.1-mouse C-region IgG1(a) autoantibody. Chimeric human-mouse IgG1(a) autoantibody colocalized with tyrosine hydroxylase in the basal ganglia within dopaminergic neurons in vivo in VH 24.3.1 Tg mice. Both human mAb 24.3.1 and IgG1(a) in Tg sera were found to react with human dopamine D2 receptor (D2R). Reactivity of chorea-derived mAb 24.3.1 or SC IgG with D2R was confirmed by dose-dependent inhibitory signaling of D2R as a potential consequence of targeting dopaminergic neurons, reaction with surface-exposed FLAG epitope-tagged D2R, and blocking of Ab reactivity by an extracellular D2R peptide. IgG from SC and a related subset of streptococcal-associated behavioral disorders called "pediatric autoimmune neuropsychiatric disorder associated with streptococci" (PANDAS) with small choreiform movements reacted in ELISA with D2R. Reaction with FLAG-tagged D2R distinguished SC from PANDAS, whereas sera from both SC and PANDAS induced inhibitory signaling of D2R on transfected cells comparably to dopamine. In this study, we define a mechanism by which the brain may be altered by Ab in movement and behavioral disorders.

Cutting edge: cardiac myosin activates innate immune responses through TLRs.

Autoimmune attack on the heart is linked to host immune responses against cardiac myosin, the most abundant protein in the heart. Although adaptive immunity is required for disease, little is known about innate immune mechanisms. In this study we report that human cardiac myosin (HCM) acted as an endogenous ligand to directly stimulate human TLRs 2 and 8 and to activate human monocytes to release proinflammatory cytokines. In addition, pathogenic epitopes of human cardiac myosin, the S2 fragment peptides S2-16 and S2-28, stimulated TLRs directly and activated human monocytes. Our data suggest that cardiac myosin and its pathogenic T cell epitopes may link innate and adaptive immunity in a novel mechanism that could promote chronic inflammation in the myocardium.

Tubulin is a neuronal target of autoantibodies in Sydenham's chorea.

Sydenham's chorea is a CNS disorder and sequela of group A streptococcal infection where deposition of Abs in brain may result in movement and neuropsychiatric abnormalities. We studied human mAbs 24.3.1, 31.1.1, and 37.2.1 derived from chorea and selected for cross-reactivity with group A streptococci and brain Ags. Our novel findings reveal that Sydenham's chorea mAbs target a 55-kDa brain protein with an N-terminal amino acid sequence of MREIVHLQ corresponding to beta-tubulin. Chorea mAb specificity for purified brain tubulin was confirmed in ELISA and Western immunoblot, and significant levels of anti-tubulin IgG were found in acute chorea sera and cerebrospinal fluid. Lysoganglioside G(M1) inhibited binding of chorea mAbs to tubulin and mAb reactivity with human caudate and putamen brain sections was blocked by anti-tubulin mAb. The chorea mAbs labeled both intra- and extracellular Ags of a neuronal cell line providing evidence suggesting mimicry between intracellular brain protein tubulin and extracellular lysoganglioside. In addition, chorea mAb 24.3.1 and acute chorea sera induced calcium/calmodulin-dependent protein kinase II activity in human neuronal cells. Nucleotide sequence analysis of the chorea mAb V(H) genes revealed that mAb 24.3.1 V(H) gene was encoded by the V(H)1 germline gene family which encodes other anti-ganglioside V(H) genes associated with motor neuropathies. mAb recognition of tubulin and the neuronal cell surface with initiation of cell signaling and dopamine release supports an emerging theme in autoimmunity whereby cross-reactive or polyreactive autoantibodies against intracellular Ags recognize cell surface epitopes potentially leading to disease.

PITX2 isoform-specific regulation of atrial natriuretic factor expression: synergism and repression with Nkx2.5.

PITX2 and Nkx2.5 are two of the earliest known transcriptional markers of vertebrate heart development. Pitx2-/- mice present with severe cardiac malformations and embryonic lethality, demonstrating a role for PITX2 in heart development. However, little is known about the downstream targets of PITX2 in cardiogenesis. We report here that the atrial natriuretic factor (ANF) promoter is a target of PITX2. PITX2A, PITX2B, and PITX2C isoforms differentially activate the ANF promoter. However, only PITX2C can synergistically activate the ANF promoter in the presence of Nkx2.5. We further demonstrate that the procollagen lysyl hydroxylase (PLOD1) promoter is regulated by Nkx2.5. Mechanistically, PITX2C and Nkx2.5 synergistically regulate ANF and PLOD1 expression through binding to their respective DNA elements. Surprisingly, PITX2A activation of the ANF and PLOD1 promoters is repressed by co-transfection of Nkx2.5 in the C3H10T1/2 embryonic fibroblast cell line. Pitx2a and Pitx2c are endogenously expressed in C3H10T1/2 cells, and these cells express factors that differentially regulate PITX2 isoform activities. We provide a new mechanism for the regulation of heart development by PITX2 isoforms through the regulation of ANF and PLOD1 gene expression and Nkx2.5 transcriptional activity.

A molecular basis for differential developmental anomalies in Axenfeld-Rieger syndrome.

Pitx2, a bicoid-like homeodomain transcription factor and Dlx2 are two transcriptional markers observed during early tooth development. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 30-fold in Chinese hamster ovary cells. Mutations in PITX2 associated with Axenfeld-Rieger syndrome (ARS) provided the first link of this homeodomain transcription factor to tooth development. We are investigating the molecular basis of developmental anomalies associated with human PITX2 mutations. A phenotypically less severe ARS mutant (without tooth anomalies), PITX2 R84W, has a similar DNA binding specificity compared to wild-type PITX2 and transactivates the Dlx2 promoter. This mutation is associated with iris hypoplasia (IH); in contrast a Rieger syndrome mutation, PITX2 T68P, which presents clinically with the full spectrum of developmental anomalies (including tooth anomalies), is unable to transactivate the Dlx2 promoter. Since Dlx2 expression is required for tooth and craniofacial development the lack of tooth anomalies in the patient with IH may be due to the residual activity of this mutant in activating the Dlx2 promoter. We demonstrate that PITX2 phosphorylation increases PITX2 and PITX2 R84W DNA binding. The PITX2 T68P ARS mutation occurs at a protein kinase C phosphorylation site in the homeodomain. Surprisingly, phosphorylation of PITX2 T68P is increased compared to wild-type PITX2 but has little effect on its DNA binding activity. Altogether these data suggest a molecular mechanism for tooth development involving Dlx2 gene expression in ARS patients.

Differential regulation of gene expression by PITX2 isoforms.

Three major PITX2 isoforms are differentially expressed in human, mice, zebrafish, chick, and frog tissues. To demonstrate differential regulation of gene expression by these isoforms we used three different promoters and three cell lines. Transient transfection of Chinese hamster ovary, HeLa, and LS-8 cell lines revealed differences in PITX2A and PITX2C activation of the PLOD1 and Dlx2 promoters, however, PITX2B is inactive. In contrast, PITX2B actives the pituitary-specific Prolactin promoter at higher levels than either PITX2A or PITX2C. Interestingly, co-transfection of either PITX2A or PITX2C with PITX2B results in a synergistic activation of the PLOD1 and Dlx2 promoters. Furthermore, PITX2 isoforms have different transcriptional activity dependent upon the cells used for transfection analysis. We have isolated a fourth PITX2 isoform (PITX2D) expressed only in humans, which acts to suppress the transcriptional activity of the other PITX2 isoforms. Electrophoretic mobility shift assays and glutathione S-transferase pull-down experiments demonstrated that all isoforms interact with PITX2D and that PITX2B forms heterodimeric complexes with PITX2A and PITX2C. Our research provides a molecular basis for differential gene regulation through the expression of PITX2 isoforms. PITX2 isoform activities are both promoter- and cell-specific, and our data reveal new mechanisms for PITX2-regulated gene expression.