Search

While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4(+) autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I-restricted epitope for PDC-E2, namely amino acid 159-167, a region very similar to the epitope recognized by MHC class II-restricted CD4(+) cells and by autoantibodies. The effector functions of these PDC-E2(159-167)-specific CD8(+) cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2(159-167)-specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2(159-167) tetramer-positive cells is heterogeneous with respect to IFN-gamma synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8(+) T cells in the PBC liver, suggesting that CD8(+) T cells play a significant role in the immunopathogenesis of PBC.

UnlabelledThe interleukin (IL)-12/IL-23-mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL-12p70, IL-12p35, interferon-gamma (IFN-γ), IL-12RB2, IL-23p40, IL-23p19, IL-17, and IL-23R using liver from PBC (n = 51) and non-PBC (n = 80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining was detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most important, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN-γ expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC. Furthermore, localized IL-23p19 production by hepatocytes may enhance profibrotic Th17 signaling and proinflammatory IFN-γ production that contribute to PBC pathology.ConclusionOur data emphasize the pathogenic relevance of IL-12/Th1 and IL-23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL-23/Th17 pathway as a potential target for therapeutic intervention.

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial Abs (AMA). The autoantigens recognized by AMA are the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxoacid dehydrogenase complex E (BCOADC-E2), and the 2-oxoglutarate dehydrogenase complex E (OGDC-E2). Previous studies using murine monoclonal and human combinatorial Abs to PDC-E2 have demonstrated an intense linear staining pattern in the apical region of biliary epithelial cells (BEC) in PBC but not control liver. We therefore examined whether mAbs to the other mitochondrial autoantigens BCOADC-E2 and OGDC-E2 demonstrated disease-specific patterns of reactivity. Using an expressed recombinant “trihybrid” protein containing the lipoyl domains of PDC-E2, OGDC-E2, and BCOADC-E2, we immunized BALB/c mice to produce 35 mAbs specific for one or more of the above mitochondrial autoantigens. Seven of these mAbs uniquely stained the apical region of BEC in PBC. Of these seven, one was reactive to PDC-E2, two recognized BCOADC-E2, three were reactive to OGDC-E2, and one recognized all three Ags. Our current data demonstrate that, similar to our previous studies regarding PDC-E2, mAbs to BCOADC-E2 and OGDC-E2, or a molecule that cross-reacts with the inner lipoyl domain of all three enzymes, also show a uniquely intense staining pattern in the apical region of BEC in patients with PBC when compared with diseased controls. The abundance of such disease-specific determinants in the target cells of PBC raises interesting possibilities regarding the role of these autoantigens in the pathogenesis of this disease.

Patients with PBC produce a directed, specific response to a single immunodominant autoepitope of PDC-E2 within the inner lipoyl domain. In contrast, immunized animals react to multiple epitopes and rarely recognize the inner lipoyl domain. In other autoimmune diseases, apoptosis plays a critical role in antigen presentation; the caspases and granzyme B are the key proteases in the generation of autoepitopes. To determine the specific cleavage pattern of full-length recombinant PDC-E2, we performed in vitro digestion with caspases-3, -6, -8 and granzyme B. The resulting fragments were immunoblotted and probed with an extensive panel of monoclonal anti-PDC-E2 antibodies and sera from patients with PBC. Interestingly, on granzyme B digestion, PDC-E2 lost reactivity, suggesting the destruction of the immunodominant epitope. Because this site contains the major epitope for both B cells and T cells, it suggests that granzyme B is unlikely to be involved in generation of autoepitopes in primary biliary cirrhosis (PBC). In contrast, following treatment with the caspase enzymes, immunoreactive fragments were generated. Indeed, by confocal microscopy, activated caspase-3 is found in the marginal hepatocytes and bile ducts. Moreover, caspase-3 staining was strongest in the small intrahepatic bile ducts, the major site of tissue destruction in PBC. In conclusion, these data suggest that following apoptosis, the caspase family of proteolytic enzymes have the potential to generate immunogenic fragments that contribute to the autoantigen reservoir and the production of antimitochondrial antibodies. These findings are also consistent with the generation of an autoimmune response against an intracellular antigen that evades catabolism during apoptosis.

Aging and certain viral infections can negatively impact humoral responses in humans. To further develop the nonhuman primate (NHP) model for investigating B cell dynamics in human aging and infectious disease, a flow cytometric panel was developed to characterize circulating rhesus B cell subsets. Significant differences between human and macaque B cells included the proportions of cells within IgD+ and switched memory populations and a prominent CD21-CD27+ unswitched memory population detected only in macaques. We then utilized the expanded panel to analyze B cell alterations associated with aging and acute simian immunodeficiency virus (SIV) infection in the NHP model. In the aging study, distinct patterns of B cell subset frequencies were observed for macaques aged one to five years compared to those between ages 5 and 30 years. In the SIV infection study, B cell frequencies and absolute number were dramatically reduced following acute infection, but recovered within four weeks of infection. Thereafter, the frequencies of activated memory B cells progressively increased; these were significantly correlated with the magnitude of SIV-specific IgG responses, and coincided with impaired maturation of anti-SIV antibody avidity, as previously reported for HIV-1 infection. These observations further validate the NHP model for investigation of mechanisms responsible for B cells alterations associated with immunosenescence and infectious disease.

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation. © 2013 Nagy et al.

A majority of individuals infected with human immunodeficiency virus (HIV) have inadequate access to antiretroviral therapy and ultimately develop debilitating oral infections that often correlate with disease progression. Due to the impracticalities of conducting host-microbe systems-based studies in HIV infected patients, we have evaluated the potential of simian immunodeficiency virus (SIV) infected rhesus macaques to serve as a non-human primate model for oral manifestations of HIV disease. We present the first description of the rhesus macaque oral microbiota and show that a mixture of human commensal bacteria and "macaque versions" of human commensals colonize the tongue dorsum and dental plaque. Our findings indicate that SIV infection results in chronic activation of antiviral and inflammatory responses in the tongue mucosa that may collectively lead to repression of epithelial development and impact the microbiome. In addition, we show that dysbiosis of the lingual microbiome in SIV infection is characterized by outgrowth of Gemella morbillorum that may result from impaired macrophage function. Finally, we provide evidence that the increased capacity of opportunistic pathogens (e.g. E. coli) to colonize the microbiome is associated with reduced production of antimicrobial peptides.

Vaginal inoculation of rhesus macaques (RM) with simian immunodeficiency virus (SIV) has been used to study the biology of HIV transmission. Although the results of vaginal SIV transmission experiments could be affected by vaginal inflammation, studies to date have been conducted without regard to levels of pre-existing genital inflammation present in RM. We collected cevicovaginal secretions (CVS) from 33-36 RM during the mid menstrual cycle (day 10-20) at 2 time points approximately 8 months apart and characterized the mRNA and protein levels of inflammatory cytokines, chemokines and interferon-stimulated genes. There was extreme variability in the levels of inflammatory mediators (IFN-α, IFN-γ, IL-6, TNF, IL-1b, IP-10, MIG, IL-12 and IL-17). In most animals, the mRNA levels of the inflammatory mediators were similar in the 2 CVS samples collected 8 months apart, suggesting that genital inflammation is stable in a subset of captive female RM. At both time points the cervicovaginal microbiota had low levels of Lactobacillus and was relatively diverse with an average of 13 genera in the samples from the first time point (median 13, range 7-21) and an average of 11.5 genera in the samples from the second time point (median 11, range 5-20). Many of the macaques had similar microbiota in the samples collected 8 months apart. However, we found no correlation between specific bacterial genera and the mRNA or protein levels of the inflammatory mediators in the genital tract of RM in this study. It seems likely that results of published vaginal SIV transmission experiments in RM have been influenced by pre-existing inflammation in the animals used for the experiments.

Author(s): Lee, Sulggi A; Bacchetti, Peter; Chomont, Nicolas; Fromentin, Remi; Lewin, Sharon R; O'Doherty, Una; Palmer, Sarah; Richman, Douglas D; Siliciano, Janet D; Yukl, Steven A; Deeks, Steven G; Burbelo, Peter D | Abstract: BackgroundA major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir"). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART).MethodsWe evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables.ResultsAmong 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of Pl0.00045. Multivariate models including age and duration of ART did not markedly alter results.ConclusionsOur findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.

BackgroundConsiderable evidence has accumulated that multiple viruses, bacteria, and protozoa manipulate interleukin-10 (IL-10)-mediated signaling through the IL-10 receptor (IL-10R) in ways that could enable establishment of a persistent microbial infection. This suggests that inhibition of pathogen targeting of IL-10/IL-10R signaling could prevent microbial persistence. Human cytomegalovirus (HCMV) and rhesus cytomegalovirus (RhCMV) express a viral interleukin-10 (cmvIL-10 and rhcmvIL-10, respectively) with comparable immune modulating properties in vitro to that of their host's cellular IL-10 (cIL-10). A prior study noted that rhcmvIL-10 alters innate and adaptive immunity to RhCMV in vivo, consistent with a central role for rhcmvIL-10 during acute virus-host interactions. Since cmvIL-10 and rhcmvIL-10 are extremely divergent from the cIL-10 of their respective hosts, vaccine-mediated neutralization of their function could inhibit establishment of viral persistence without inhibition of cIL-10.Methods and findingsAs a prelude to evaluating cmvIL-10-based vaccines in humans, the rhesus macaque model of HCMV was used to interrogate peripheral and mucosal immune responses to rhcmvIL-10 in RhCMV-infected animals. ELISA were used to detect rhcmvIL-10-binding antibodies in plasma and saliva, and an IL-12-based bioassay was used to quantify plasma antibodies that neutralized rhcmvIL-10 function. rhcmvIL-10 is highly immunogenic during RhCMV infection, stimulating high avidity rhcmvIL-10-binding antibodies in the plasma of all infected animals. Most infected animals also exhibited plasma antibodies that partially neutralized rhcmvIL-10 function but did not cross-neutralize the function of rhesus cIL-10. Notably, minimally detectable rhcmvIL-10-binding antibodies were detected in saliva.ConclusionThis study demonstrates that rhcmvIL-10, as a surrogate for cmvIL-10, is a viable vaccine candidate because (1) it is highly immunogenic during natural RhCMV infection, and (2) neutralizing antibodies to rhcmvIL-10 do not cross-react with rhesus cIL-10. Exceedingly low rhcmvIL-10 antibodies in saliva further suggest that the oral mucosa, which is critical in RhCMV natural history, is associated with suboptimal anti-rhcmvIL-10 antibody responses.