Regulatory T cells limit age-associated retinal inflammation and neurodegeneration.

BACKGROUND: Ageing is the principal risk factor for retinal degenerative diseases, which are the commonest cause of blindness in the developed countries. These conditions include age-related macular degeneration or diabetic retinopathy. Regulatory T cells play a vital role in immunoregulation of the nervous system by limiting inflammation and tissue damage in health and disease. Because the retina was long-considered an immunoprivileged site, the precise contribution of regulatory T cells in retinal homeostasis and in age-related retinal diseases remains unknown. METHODS: Regulatory T cells were selectively depleted in both young (2-4 months) and aged (18-23 months) FoxP3-DTR mice. We evaluated neuroretinal degeneration, gliosis, subretinal space phagocyte infiltration, and retinal pigmented epithelium morphology through immunofluorescence analysis. Subsequently, aged Treg depleted animals underwent adoptive transfer of both young and aged regulatory T cells from wild-type mice, and the resulting impact on neurodegeneration was assessed. Statistical analyses employed included the U-Mann Whitney test, and for comparisons involving more than two groups, 1-way ANOVA analysis followed by Bonferroni's post hoc test. RESULTS: Our study shows that regulatory T cell elimination leads to retinal pigment epithelium cell dysmorphology and accumulation of phagocytes in the subretinal space of young and aged mice. However, only aged mice experience retinal neurodegeneration and gliosis. Surprisingly, adoptive transfer of young but not aged regulatory T cells reverse these changes. CONCLUSION: Our findings demonstrate an essential role for regulatory T cells in maintaining age retinal homeostasis and preventing age-related neurodegeneration. This previously undescribed role of regulatory T cells in limiting retinal inflammation, RPE/choroid epithelium damage and subsequently photoreceptor loss with age, opens novel avenues to explore regulatory T cell neuroprotective and anti-inflammatory properties as potential therapeutic approaches for age-related retinal diseases.

Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination.

Myelin regeneration (remyelination) is essential to prevent neurodegeneration in demyelinating diseases such as Multiple Sclerosis, however, its efficiency declines with age. Regulatory T cells (Treg) recently emerged as critical players in tissue regeneration, including remyelination. However, the effect of ageing on Treg-mediated regenerative processes is poorly understood. Here, we show that expansion of aged Treg does not rescue age-associated remyelination impairment due to an intrinsically diminished capacity of aged Treg to promote oligodendrocyte differentiation and myelination in male and female mice. This decline in regenerative Treg functions can be rescued by a young environment. We identified Melanoma Cell Adhesion Molecule 1 (MCAM1) and Integrin alpha 2 (ITGA2) as candidates of Treg-mediated oligodendrocyte differentiation that decrease with age. Our findings demonstrate that ageing limits the neuroregenerative capacity of Treg, likely limiting their remyelinating therapeutic potential in aged patients, and describe two mechanisms implicated in Treg-driven remyelination that may be targetable to overcome this limitation.

Regulation of caries-induced pulp inflammation by NLRP3 inflammasome: A laboratory-based investigation.

AIM: To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. METHODOLOGY: NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP-1 macrophages expressing the apoptosis-related speck-like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex-vivo pulpitis model in which the DPCs were co-cultured with THP-1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤ .05. RESULTS: NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 ß-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-ß release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p < .01). NLRP3-specific inhibitor, MCC950, significantly reduced IL-1ß and IL-6 in an ex-vivo pulpitis model (p < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). CONCLUSIONS: Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries-induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation.

Innate and adaptive immune mechanisms regulating central nervous system remyelination.

Neuroinflammation is a hallmark of many neurodegenerative diseases and is considered their underlying cause. However, certain aspects of neuroinflammation favour beneficial outcomes after damage including the regeneration of myelin (remyelination). Both innate and adaptive immune mechanisms have been recognised as central to remyelination success. In particular, central nervous system (CNS) microglia and macrophages are established as key regulators of remyelination in the injured CNS with recently discovered novel mechanisms that underpin remyelination. How the adaptive immune system contributes to and regulates remyelination, however, is less established. Owing to their immunomodulatory and recently discovered proregenerative functions including in the CNS, regulatory T cells were identified as key for successful remyelination, but many gaps in the underlying mechanisms remain. As there are no therapies yet that enhance remyelination after damage, harnessing the beneficial aspects of neuroinflammation could underpin proregenerative CNS therapies of the future.

Evidence That a TRPA1-Mediated Murine Model of Temporomandibular Joint Pain Involves NLRP3 Inflammasome Activation.

This study investigates the role of transient receptor potential ankyrin 1 (TRPA1) in murine temporomandibular joint (TMJ) inflammatory hyperalgesia and the influence of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. Two distinct murine models of TMJ pain and inflammation (zymosan and CFA) were established. Spontaneous pain-like behaviours were observed as unilateral front paw cheek wipes. Ipsilateral cheek blood flow was used as a measure of ongoing inflammation, which, to our knowledge, is a novel approach to assessing real-time inflammation in the TMJ. Joint tissue and trigeminal ganglia were collected for ex vivo investigation. Both zymosan and CFA induced a time-dependent increase in hyperalgesia and inflammation biomarkers. Zymosan induced a significant effect after 4 h, correlating with a significantly increased IL-1ß protein expression. CFA (50 µg) induced a more sustained response. The TRPA1 receptor antagonist A967079 significantly inhibited hyper-nociception. The NLRP3 inhibitor MCC950 similarly inhibited hyper-nociception, also attenuating inflammatory markers. In the trigeminal ganglia, CFA-induced CGRP expression showed trends of inhibition by A967079, whilst lba1 immunofluorescence was significantly inhibited by A967079 and MCC950, where the effect of TRPA1 inhibition lasted up to 14 days. Our results show that stimulation of TRPA1 is key to the TMJ pain. However, the inflammasome inhibitor exhibited similar properties in attenuating these pain-like behaviours, in addition to some inflammatory markers. This indicates that in addition to the therapeutic targeting of TRPA1, NLRP3 inhibition may provide a novel therapeutic strategy for TMJ inflammation and pain.

The microbiota regulates murine inflammatory responses to toxin-induced CNS demyelination but has minimal impact on remyelination.

The microbiota is now recognized as a key influence on the host immune response in the central nervous system (CNS). As such, there has been some progress toward therapies that modulate the microbiota with the aim of limiting immune-mediated demyelination, as occurs in multiple sclerosis. However, remyelination-the regeneration of myelin sheaths-also depends upon an immune response, and the effects that such interventions might have on remyelination have not yet been explored. Here, we show that the inflammatory response during CNS remyelination in mice is modulated by antibiotic or probiotic treatment, as well as in germ-free mice. We also explore the effect of these changes on oligodendrocyte progenitor cell differentiation, which is inhibited by antibiotics but unaffected by our other interventions. These results reveal that high combined doses of oral antibiotics impair oligodendrocyte progenitor cell responses during remyelination and further our understanding of how mammalian regeneration relates to the microbiota.

Oligodendrocytes in Development, Myelin Generation and Beyond.

Oligodendrocytes are the myelinating cells of the central nervous system (CNS) that are generated from oligodendrocyte progenitor cells (OPC). OPC are distributed throughout the CNS and represent a pool of migratory and proliferative adult progenitor cells that can differentiate into oligodendrocytes. The central function of oligodendrocytes is to generate myelin, which is an extended membrane from the cell that wraps tightly around axons. Due to this energy consuming process and the associated high metabolic turnover oligodendrocytes are vulnerable to cytotoxic and excitotoxic factors. Oligodendrocyte pathology is therefore evident in a range of disorders including multiple sclerosis, schizophrenia and Alzheimer's disease. Deceased oligodendrocytes can be replenished from the adult OPC pool and lost myelin can be regenerated during remyelination, which can prevent axonal degeneration and can restore function. Cell population studies have recently identified novel immunomodulatory functions of oligodendrocytes, the implications of which, e.g., for diseases with primary oligodendrocyte pathology, are not yet clear. Here, we review the journey of oligodendrocytes from the embryonic stage to their role in homeostasis and their fate in disease. We will also discuss the most common models used to study oligodendrocytes and describe newly discovered functions of oligodendrocytes.

Regenerating CNS myelin: Emerging roles of regulatory T cells and CCN proteins.

Efficient myelin regeneration in the central nervous system (CNS) requires the migration, proliferation and differentiation of oligodendrocyte progenitor cells (OPC) into myelinating oligodendrocytes. In demyelinating diseases such as multiple sclerosis (MS), this regenerative process can fail, and therapies targeting myelin repair are currently completely lacking in the clinic. The immune system is emerging as a key regenerative player in many tissues, such as muscle and heart. We recently reported that regulatory T cells (Treg) are required for efficient CNS remyelination. Furthermore, Treg secrete CCN3, a matricellular protein from the CCN family, implicated in regeneration of other tissues. Treg-derived CCN3 promoted oligodendrocyte differentiation and myelination. In contrast, previous studies showed that CCN2 inhibited myelination. These studies highlight the need for further scrutiny of the roles that CCN proteins play in myelin development and regeneration. Collectively, these findings open up exciting avenues of research to uncover the regenerative potential of the adaptive immune system.

Characterization of a murine mixed neuron-glia model and cellular responses to regulatory T cell-derived factors.

One of the unmet clinical needs in demyelinating diseases such as Multiple Sclerosis (MS) is to provide therapies that actively enhance the process of myelin regeneration (remyelination) in the central nervous system (CNS). Oligodendrocytes, the myelinating cells of the CNS, play a central role in remyelination and originate from oligodendrocyte progenitor cells (OPCs). We recently showed that depletion of regulatory T cells (Treg) impairs remyelination in vivo, and that Treg-secreted factors directly enhance oligodendrocyte differentiation. Here we aim to further characterize the dynamics of Treg-enhanced oligodendrocyte differentiation as well as elucidate the cellular components of a murine mixed neuron-glia model. Murine mixed neuron-glia cultures were generated from P2-7 C57BL/6 mice and characterized for percentage of neuronal and glial cell populations prior to treatment at 7 days in vitro (div) as well as after treatment with Treg-conditioned media at multiple timepoints up to 12 div. Mixed neuron-glia cultures consisted of approximately 30% oligodendroglial lineage cells, 20% neurons and 10% microglia. Furthermore, a full layer of astrocytes, that could not be quantified, was present. Treatment with Treg-conditioned media enhanced the proportion of MBP+ oligodendrocytes and decreased the proportion of PDGFRα+ OPCs, but did not affect OPC proliferation or survival. Treg-enhanced oligodendrocyte differentiation was not caused by Treg polarizing factors, was dependent on the number of activation cycles Treg underwent and was robustly achieved by using 5% conditioned media. These studies provide in-depth characterization of a murine mixed neuron-glia model as well as further insights into the dynamics of Treg-enhanced oligodendrocyte differentiation.

QuPath: Open source software for digital pathology image analysis.

QuPath is new bioimage analysis software designed to meet the growing need for a user-friendly, extensible, open-source solution for digital pathology and whole slide image analysis. In addition to offering a comprehensive panel of tumor identification and high-throughput biomarker evaluation tools, QuPath provides researchers with powerful batch-processing and scripting functionality, and an extensible platform with which to develop and share new algorithms to analyze complex tissue images. Furthermore, QuPath's flexible design makes it suitable for a wide range of additional image analysis applications across biomedical research.

Regulatory T cells promote myelin regeneration in the central nervous system.

Regeneration of CNS myelin involves differentiation of oligodendrocytes from oligodendrocyte progenitor cells. In multiple sclerosis, remyelination can fail despite abundant oligodendrocyte progenitor cells, suggesting impairment of oligodendrocyte differentiation. T cells infiltrate the CNS in multiple sclerosis, yet little is known about T cell functions in remyelination. We report that regulatory T cells (Treg) promote oligodendrocyte differentiation and (re)myelination. Treg-deficient mice exhibited substantially impaired remyelination and oligodendrocyte differentiation, which was rescued by adoptive transfer of Treg. In brain slice cultures, Treg accelerated developmental myelination and remyelination, even in the absence of overt inflammation. Treg directly promoted oligodendrocyte progenitor cell differentiation and myelination in vitro. We identified CCN3 as a Treg-derived mediator of oligodendrocyte differentiation and myelination in vitro. These findings reveal a new regenerative function of Treg in the CNS, distinct from immunomodulation. Although the cells were originally named 'Treg' to reflect immunoregulatory roles, this also captures emerging, regenerative Treg functions.

The TGF-ß-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8⁺ T cells.

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-ß are well established, we wondered whether TGF-ß could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-ß promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-ß up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-ß type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-ß, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-ß signaling.

Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris.

In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e., protective by acting against Propionibacterium acnes, pro-inflammatory by acting as signalling molecules. The cutaneous expression of 15 different AMPs was investigated in acne patients; furthermore, the impact of isotretinoin therapy on AMP expression was analysed in skin biopsies from 13 patients with acne vulgaris taken before, during and after a 6-month treatment cycle with isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous expression of the AMPs cathelicidin, human ß-defensin-2 (HBD-2), lactoferrin, lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated compared to controls. While relative expression levels of cathelicidin, HBD-2, lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during isotretinoin treatment, only those of cathelicidin and koebnerisin returned to normal after 6 months of isotretinoin therapy. The increased expression of lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and dermcidin were not regulated in untreated acne patients and isotretinoin had no effect on these AMPs. In conclusion, the expression of various AMPs is altered in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of distinct AMPs while the expression of others is still increased in healing acne. Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules could serve as specific targets for acne therapy and maintenance of clinical remission.

Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis.

Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age-related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow-derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow-derived macrophages and bone marrow monocytes did not exhibit age-related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age-related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell-derived IL-10 was increased in resting and LPS-activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue-resident peritoneal macrophages, but not by bone marrow-derived macrophages/monocytes, and suggest that age-related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.

Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment.

BACKGROUND: We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor α (TNF-α) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD). DESIGN: Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab. RESULTS: Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-γ-secreting Th1 cells and IFN-α-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab. CONCLUSIONS: New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL-23 antibody therapy is a highly effective therapy for these lesions.

Narrow-band ultraviolet B treatment boosts serum 25-hydroxyvitamin D in patients with psoriasis on oral vitamin D supplementation.

A course of treatment with narrow-band ultraviolet B (NB-UVB) improves psoriasis and increases serum 25-hydroxyvitamin D (25(OH)D). In this study 12 patients with psoriasis who were supplemented with oral cholecalciferol, 20 µg daily, were given a course of NB-UVB and their response measured. At baseline, serum 25(OH)D was 74.14 ± 22.9 nmol/l. At the 9th exposure to NB-UVB 25(OH)D had increased by 13.2 nmol/l (95% confidence interval (95% CI) 7.2-18.4) and at the 18th exposure by 49.4 nmol/l (95% CI 35.9-64.6) above baseline. Psoriasis Area Severity Index score improved from 8.7 ± 3.5 to 4.5 ± 2.0 (p < 0.001). At baseline, psoriasis lesions showed low vitamin D metabolizing enzyme (CYP27A1, CYP27B1) and high human ß-defensin-2 mRNA expression levels compared with those of the healthy subjects. In conclusion, NB-UVB treatment significantly increases serum 25(OH)D in patients with psoriasis who are taking oral vitamin D supplementation, and the concentrations remain far from the toxicity level. Healing psoriasis lesions show similar mRNA expression of vitamin D metabolizing enzymes, but higher antimicrobial peptide levels than NB-UVB-treated skin in healthy subjects.

A narrow-band ultraviolet B course improves vitamin D balance and alters cutaneous CYP27A1 and CYP27B1 mRNA expression levels in haemodialysis patients supplemented with oral vitamin D.

BACKGROUND/AIMS: Chronic kidney disease (CKD) patients on dialysis are prone to vitamin D insufficiency despite oral vitamin D supplementation. Here, we studied whether narrow-band ultraviolet B (NB-UVB) exposures improve vitamin D balance. METHODS: 14 haemodialysis patients and 15 healthy subjects receiving oral cholecalciferol 20 µg daily got nine NB-UVB exposures on the entire body. Serum 25-hydroxyvitamin D (25(OH)D) was measured by radioimmunoassay. Cutaneous mRNA expression levels of CYP27A1 and CYP27B1, two enzymes required for hydroxylation of vitamin D into its active metabolite, were also measured. RESULTS: The baseline serum 25(OH)D concentration was 57.6 ± 18.2 nmol/l in the CKD patients and 74.3 ± 14.8 nmol/l in the healthy subjects. The NB-UVB course increased serum 25(OH)D by 14.0 nmol/l (95% CI 8.7-19.5) and 17.0 nmol/l (CI 13.7-20.2), respectively. At baseline the CKD patients showed significantly increased CYP27B1 levels compared to the healthy subjects. CONCLUSIONS: A short NB-UVB course is an efficient way to improve vitamin D balance in CKD patients on dialysis who are receiving oral vitamin D supplementation. The increased cutaneous CYP27B1 levels in the CKD patients suggest that the loss of renal activity of this enzyme is at least partially compensated for by the skin.

Antimicrobial peptides and proinflammatory cytokines in periprosthetic joint infection.

BACKGROUND: Differentiation between septic and aseptic loosening of joint replacements is essential for successful revision surgery, but reliable markers for the diagnosis of low-grade infection are lacking. The present study was performed to assess intra-articular and systemic levels of antimicrobial peptides and proinflammatory cytokines as diagnostic markers for periprosthetic joint infection. METHODS: Fifteen consecutive patients with staphylococcal periprosthetic joint infections and twenty control patients with aseptic loosening of total hip and knee replacements were included in this prospective, single-center, controlled clinical trial. Expression of the antimicrobial peptides human ß-defensin-2 (HBD-2), human ß-defensin-3 (HBD-3), and cathelicidin LL-37 (LL-37) was determined by ELISA (enzyme-linked immunosorbent assay) in serum and joint aspirates. Proinflammatory cytokines were assessed in serum and joint aspirates with use of cytometric bead arrays. C-reactive protein in serum, microbiology, and histopathology of periprosthetic tissue served as the "gold standard" for the diagnosis of infection. RESULTS: The antimicrobial peptides HBD-3 and LL-37 were significantly elevated in joint aspirates from patients with periprosthetic joint infection compared with patients with aseptic loosening, and the area under the curve (AUC) in a receiver operating characteristic curve analysis was equal to 0.745 and 0.875, respectively. Additionally, significant local increases in the proinflammatory cytokines interleukin (IL)-1ß, IL-4, IL-6, IL-17A, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were observed to be associated with infection. Logistic regression analysis indicated that the combination of an antimicrobial peptide with another synovial fluid biomarker improved diagnostic accuracy; the AUC value was 0.916 for LL-37 and IL-4, 0.895 for LL-37 and IL-6, 0.972 for HBD-3 and IL-4, and 0.849 for HBD-3 and IL-6. In contrast, the only antimicrobial peptides and cytokines in serum that showed a significant systemic increase in association with infection were HBD-2, IL-4, and IL-6 (all of which had an AUC value of <0.75). CONCLUSIONS: The present study showed promising results for the use of antimicrobial peptides and other biomarkers in synovial fluid for the diagnosis of periprosthetic joint infection, and analysis of the levels in synovial fluid was more accurate than analysis of serum.