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Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function.
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The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.
Assuntos
Ácidos Docosa-Hexaenoicos , Ácidos Graxos Ômega-3 , Humanos , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Azidas , Cobre/farmacologia , Biotina/farmacologia , Leucotrieno B4/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Macrófagos , Óleos de Peixe/farmacologia , Anti-Inflamatórios/farmacologia , Alcinos/farmacologia , Prostaglandinas , OxirredutasesRESUMO
Polyunsaturated fatty acids (PUFAs) and their metabolites are potent regulators of inflammation. Generally, omega (n)-3 PUFAs are considered proresolving whereas n-6 PUFAs are classified as proinflammatory. In this study, we characterized the inflammatory response in murine peritonitis and unexpectedly found the accumulation of adrenic acid (AdA), a poorly studied n-6 PUFA. Functional studies revealed that AdA potently inhibited the formation of the chemoattractant leukotriene B4 (LTB4), specifically in human neutrophils, and this correlated with a reduction of its precursor arachidonic acid (AA) in free form. AdA exposure in human monocyte-derived macrophages enhanced efferocytosis of apoptotic human neutrophils. In vivo, AdA treatment significantly alleviated arthritis in an LTB4-dependent murine arthritis model. Our findings are, to our knowledge, the first to indicate that the n-6 fatty acid AdA effectively blocks production of LTB4 by neutrophils and could play a role in resolution of inflammation in vivo.
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Anti-Inflamatórios/metabolismo , Artrite Experimental/imunologia , Ácidos Graxos Ômega-6/metabolismo , Ácidos Graxos Insaturados/metabolismo , Peritonite/imunologia , Animais , Anti-Inflamatórios/análise , Ácido Araquidônico/metabolismo , Artrite Experimental/patologia , Células Cultivadas , Ácidos Graxos Ômega-6/análise , Ácidos Graxos Insaturados/análise , Humanos , Leucotrieno B4/metabolismo , Lipidômica , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Lavagem Peritoneal , Peritonite/patologia , Cultura Primária de Células , Células THP-1 , Zimosan/administração & dosagem , Zimosan/imunologiaRESUMO
OBJECTIVES: Antibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in the sera of rheumatoid arthritis (RA) patients. These autoantibodies associate with increased risk of RA development and with severity of joint destruction. It is not known which proteins in the RA joint are recognised by anti-CarP antibodies. Therefore, we investigated the presence and identity of carbamylated proteins in the human (inflamed) joint. METHODS: We obtained synovium, cartilage and synovial fluid from RA joints. Cartilage and synovium were obtained from controls. Samples were processed and used for immunohistochemistry or mass-spectrometric analysis to investigate the presence of carbamylated proteins. Anti-CarP antibody reactivity towards identified carbamylated proteins was tested by ELISA. RESULTS: Immunohistochemistry showed extensive staining of RA and control synovial tissue. Whole proteome analyses of the joint tissues revealed a large number of carbamylated peptidyllysine residues. We identified many carbamylated proteins in cartilage and were also able to detect carbamylation in synovial tissue and synovial fluid. Carbamylation was not exclusive to the RA joint and was also present in the joints of controls. Anti-CarP antibodies in the sera of RA patients were able to recognise the identified carbamylated proteins. CONCLUSIONS: We conclude that numerous carbamylated proteins are present in the RA joint. These carbamylated proteins can be recognised by anti-CarP antibodies, substantiating the notion that anti-CarP antibodies may play a role in the pathogenesis of RA.
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Artrite Reumatoide , Autoanticorpos , Ensaio de Imunoadsorção Enzimática , Humanos , Espectrometria de Massas , Membrana SinovialRESUMO
Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL-6-secretion by CD4+ T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL-6+ CD4+ T cells from SVF display a more activated phenotype than the IL-6- T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL-6-secreting CD4+ T cells cannot be assigned to a conventional Th subset. TCRß gene analysis revealed that IL-6+ and IL-6- CD4+ T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL-6 production by CD4+ T cells. Thus, IL-6+ CD4+ T cells are TCRαß T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint.
Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-6/metabolismo , Idoso , Linfócitos T CD4-Positivos/classificação , Feminino , Humanos , Imunofenotipagem , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/imunologia , Osteoartrite do Joelho/patologia , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologiaRESUMO
Previous studies have shown accumulation and an enhanced proinflammatory profile of macrophages in adipose tissue of obese mice, indicating the presence of an interaction between adipocytes and macrophages in this tissue. However, the consequences of this interaction in humans are yet incompletely understood. In this study, we explored the modulating effects of adipocytes on the phenotype of macrophages in humans and studied the possible molecular pathways involved. Adipocyte-conditioned media (ACM) treatment of macrophages for 48 h strongly reduced the LPS-induced IL-12p40 secretion by macrophages, whereas the production of TNF-α and other cytokines remained largely unaffected. This effect was independent of the source of adipocytes. Interestingly, the level of inhibition correlated directly with body mass index (BMI) of the adipocyte donor. Because adipocytes release many different cytokines, adipokines, and lipids, we have separated the protein and lipid fractions of ACM, to obtain insight into the molecular nature of the soluble mediators underlying the observed effect. These experiments revealed that the inhibitory effect resided predominantly in the lipid fraction. Further studies revealed that PGE2 and linoleic and oleic acid were potent inhibitors of IL-12p40 secretion. Interestingly, concentrations of these ACM-derived lipids increased with increase in BMI of the adipocyte donor, suggesting that they could mediate the BMI-dependent effects of ACM. To our knowledge, these results provide first evidence that obesity-related changes in adipose tissue macrophage phenotype could be mediated by adipocyte-derived lipids in humans. Intriguingly, these changes appear to be different from those in murine obesity.
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Adipócitos/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Macrófagos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Índice de Massa Corporal , Comunicação Celular/imunologia , Meios de Cultivo Condicionados , Dinoprostona/metabolismo , Humanos , Ácido Linoleico/metabolismo , Lipídeos , Lipopolissacarídeos , Macrófagos/imunologia , Obesidade/metabolismo , Ácido Oleico/metabolismo , Fenótipo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Adipose tissue contains several immune cells whose number and phenotype vary depending on the adiposity. In the present study, we show that IFN-γ(+) CD4(+) T cells are enriched in human adipose tissue compared with in blood. To gain insight into the underlying mechanisms, we investigated the possibility that human adipocytes modulate CD4(+) T-cell cytokine production and proliferation and show that CD4(+) T cells produced increased levels of IFN-γ when activated in the presence of adipocytes. This effect was mediated by soluble mediators, as shown in transwell and adipocyte-conditioned medium (ACM) transfer experiments. Additionally, ACM induced increased proliferation of CD4(+) T cells upon activation. Intriguingly, the proliferation-enhancing effect resided mainly in the lipid fraction of ACM, as shown upon separation of the protein and lipid fraction. Further separation of these lipids based on polarity revealed that the modulatory effect is confined to fractions containing free fatty acids. All identified fatty acids were able to individually enhance T-cell proliferation. These data indicate that adipocytes can modulate CD4(+) T-cell function through the release of lipids. Remarkably, free fatty acids were the most prominent modulators of T-cell proliferation, possibly leading to an accumulation of these cells in adipose tissue.
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Adipócitos/imunologia , Linfócitos T CD4-Positivos/imunologia , Ácidos Graxos não Esterificados/imunologia , Obesidade/imunologia , Adipócitos/patologia , Tecido Adiposo/patologia , Comunicação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , Ácidos Graxos não Esterificados/isolamento & purificação , Humanos , Imunomodulação , Interferon gama/imunologiaRESUMO
T cells are the most common immune cells in atherosclerotic plaques, and the function of T cells can be altered by fatty acids. Here, we show that pre-exposure of CD4+ T cells to oleic acid, an abundant fatty acid linked to cardiovascular events, upregulates core metabolic pathways and promotes differentiation into interleukin-9 (IL-9)-producing cells upon activation. RNA sequencing of non-activated T cells reveals that oleic acid upregulates genes encoding key enzymes responsible for cholesterol and fatty acid biosynthesis. Transcription footprint analysis links these expression changes to the differentiation toward TH9 cells, a pro-atherogenic subset. Spectral flow cytometry shows that pre-exposure to oleic acid results in a skew toward IL-9+-producing T cells upon activation. Importantly, pharmacological inhibition of either cholesterol or fatty acid biosynthesis abolishes this effect, suggesting a beneficial role for statins beyond cholesterol lowering. Taken together, oleic acid may affect inflammatory diseases like atherosclerosis by rewiring T cell metabolism.
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OBJECTIVE: Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA). METHODS: E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA. RESULTS: Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins. CONCLUSIONS: Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.
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Linfócitos B , Animais , Camundongos , Acetilação , Humanos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Escherichia coli/imunologia , Proteínas de Bactérias/imunologia , Reações Cruzadas/imunologia , Formação de Anticorpos/imunologia , Camundongos Endogâmicos C57BL , Antígenos de Bactérias/imunologia , Artrite Reumatoide/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/imunologiaAssuntos
Adipocinas/metabolismo , Tecido Adiposo/patologia , Artrite Reumatoide/patologia , Osteoartrite do Joelho/patologia , Sinovite/patologia , Tecido Adiposo/fisiopatologia , Idoso , Artrite Reumatoide/fisiopatologia , Biópsia por Agulha , Feminino , Humanos , Imuno-Histoquímica , Articulação do Joelho/fisiopatologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/fisiopatologia , Patela/patologia , Patela/fisiopatologia , Prognóstico , Sinovite/fisiopatologiaRESUMO
Many autoimmune diseases (AIDs) are characterized by the persistence of autoreactive B cell responses, which have been directly implicated in disease pathogenesis. How and why these cells are generated or how they are maintained for years is largely unknown. Rheumatoid arthritis (RA) is among the most common AIDs and is characterized by autoantibodies recognizing proteins with posttranslational modifications (PTMs). This PTM-directed autoreactive B cell compartment is ill defined. Here, we visualized the B cell response against the three main types of PTM antigens implicated in RA by spectral flow cytometry. Our results showed extensive cross-reactivity of PTM-directed B cells against all three PTM antigens (citrulline, homocitrulline, and acetyllysine). Unsupervised clustering revealed several distinct memory B cell (mBC) populations. PTM-directed cells clustered with the most recently activated class-switched mBC phenotype, with high CD80, low CD24, and low CD21 expression. Notably, patients also harbored large fractions of PTM-directed plasmablasts (PBs). Both PTM-directed mBCs and PBs showed high expression of CXCR3, a receptor for chemokines present in abundance in arthritic joints. Together, our data provide detailed insight into the biology of B cell autoreactivity and its remarkable, seemingly exhaustless persistence in a prominent human AID.
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Artrite Reumatoide , Células B de Memória , Humanos , Linfócitos B , Plasmócitos , Autoanticorpos , Antígenos , Receptores CXCR3RESUMO
OBJECTIVE: Adipose tissue can secrete soluble mediators (adipokines) with potent immune regulatory functions. Some adipokines have been previously associated with radiographic damage in patients with rheumatoid arthritis (RA). In the present study, we investigated the capacity of baseline adipokine levels to predict radiographic progression over a period of 4 years and studied their contribution relative to that of other known risk factors, such as anti-cyclic citrullinated peptide (anti-CCP) antibodies. METHODS: Serum concentrations of leptin, visfatin, resistin, adiponectin, adipsin, tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) were determined in serum samples obtained at baseline from 253 patients with RA from the Early Arthritis Cohort. The association between levels of these adipokines and radiographic progression was determined using a multivariate normal regression model correcting for age, sex, treatment strategy, body mass index (BMI), and the presence of anti-CCP antibodies. RESULTS: Levels of IL-6, TNFα, visfatin, and adiponectin were positively associated with radiographic progression over 4 years. This association was independent of BMI. However, only adiponectin levels remained significantly associated with radiographic progression when the model was corrected for the presence of anti-CCP antibodies, whereas a trend was observed for IL-6. The association of both TNFα and visfatin with radiographic damage disappeared after correction for the presence of anti-CCP antibodies, which is consistent with the fact that the levels of both cytokines correlated significantly with anti-CCP antibody levels in these patients. CONCLUSION: Our results indicate that adipokines are predictors of radiographic progression in RA, possibly through distinct underlying biologic mechanisms.
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Adipocinas/sangue , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico por imagem , Progressão da Doença , Adulto , Idoso , Autoanticorpos/sangue , Biomarcadores/sangue , Feminino , Pé/diagnóstico por imagem , Mãos/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos Cíclicos/sangue , Peptídeos Cíclicos/imunologia , Valor Preditivo dos Testes , RadiografiaRESUMO
The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.
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BACKGROUND: Obesity is associated with the development and progression of osteoarthritis (OA). Although the infrapatellar fat pad (IFP) could be involved in this association, due to its intracapsular localization in the knee joint, there is currently little known about the effect of obesity on the IFP. Therefore, we investigated cellular and molecular body mass index (BMI)-related features in the IFP of OA patients. METHODS: Patients with knee OA (N = 155, 68% women, mean age 65 years, mean (SD) BMI 29.9 kg/m2 (5.7)) were recruited: IFP volume was determined by magnetic resonance imaging in 79 patients with knee OA, while IFPs and subcutaneous adipose tissue (SCAT) were obtained from 106 patients undergoing arthroplasty. Crown-like structures (CLS) were determined using immunohistochemical analysis. Adipocyte size was determined by light microscopy and histological analysis. Stromal vascular fraction (SVF) cells were characterized by flow cytometry. RESULTS: IFP volume (mean (SD) 23.6 (5.4) mm3) was associated with height, but not with BMI or other obesity-related features. Likewise, volume and size of IFP adipocytes (mean 271 pl, mean 1933 µm) was not correlated with BMI. Few CLS were observed in the IFP, with no differences between overweight/obese and lean individuals. Moreover, high BMI was not associated with higher SVF immune cell numbers in the IFP, nor with changes in their phenotype. No BMI-associated molecular differences were observed, besides an increase in TNFα expression with high BMI. Macrophages in the IFP were mostly pro-inflammatory, producing IL-6 and TNFα, but little IL-10. Interestingly, however, CD206 and CD163 were associated with an anti-inflammatory phenotype, were the most abundantly expressed surface markers on macrophages (81% and 41%, respectively) and CD163+ macrophages had a more activated and pro-inflammatory phenotype than their CD163- counterparts. CONCLUSIONS: BMI-related features usually observed in SCAT and visceral adipose tissue could not be detected in the IFP of OA patients, a fat depot implicated in OA pathogenesis.
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Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Índice de Massa Corporal , Macrófagos/metabolismo , Osteoartrite do Joelho/metabolismo , Patela/metabolismo , Tecido Adiposo/diagnóstico por imagem , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/metabolismo , Imageamento por Ressonância Magnética , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/diagnóstico por imagem , Obesidade/metabolismo , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/diagnóstico por imagem , Patela/diagnóstico por imagem , Receptores de Superfície Celular/metabolismoRESUMO
OBJECTIVE: To get a better understanding of inflammatory pathways active in the osteoarthritic (OA) joint, we characterized and compared inflammatory cells in the synovium and the infrapatellar fat pad (IFP) of patients with knee OA. METHODS: Infiltrating immune cells were characterized by flow cytometry in 76 patients with knee OA (mean age 63.3, 52% women, median body mass index 28.9) from whom synovial tissue (n = 40) and IFP (n = 68) samples were obtained. Pain was assessed by the visual analog scale (VAS; 0-100 mm). Spearman rank correlations and linear regression analyses adjusted for sex and age were performed. RESULTS: Macrophages and T cells, followed by mast cells, were the most predominant immune cells in the synovium and IFP, and were equally abundant in these tissues. Macrophages and T cells secreted mostly proinflammatory cytokines even without additional stimulation, indicating their activated state. Accordingly, most CD4+ T cells had a memory phenotype and contained a significant population of cells expressing activation markers (CD25+, CD69+). Interestingly, the percent of CD69+ T cells was higher in synovial than IFP CD4+ T cells. Preliminary analyses indicated that the number of synovial CD4+ T cells were associated with VAS pain (ß 0.51, 95% CI 0.09-1.02, p = 0.02). CONCLUSION: Our data suggest that the immune cell composition of the synovium and the IFP is similar, and includes activated cells that could contribute to inflammation through secretion of proinflammatory cytokines. Moreover, preliminary analyses indicate that synovial CD4+ T cells might associate with pain in patients with endstage OA of the knee.
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Tecido Adiposo/patologia , Articulação do Joelho/patologia , Macrófagos/patologia , Osteoartrite do Joelho/patologia , Membrana Sinovial/patologia , Linfócitos T/patologia , Tecido Adiposo/metabolismo , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Articulação do Joelho/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/metabolismo , Membrana Sinovial/metabolismo , Linfócitos T/metabolismoRESUMO
OBJECTIVE: To investigate whether high molecular weight adiponectin (hmwAPN) mediates the associations of total adiponectin (totAPN) with radiographic progression in rheumatoid arthritis (RA) and hand osteoarthritis (HOA). METHODS: Associations between baseline hmwAPN or totAPN levels with radiographic progression were determined using multivariate linear regression or generalized estimated equations. RESULTS: In patients with RA, totAPN associated positively, whereas in patients with HOA it associated negatively with radiographic progression. In contrast, hmwAPN did not associate significantly with radiographic progression in either cohort. CONCLUSION: Our data indicate that the differential effects associated between totAPN and radiographic progression in either RA or HOA are not mediated by hmwAPN.