Abstract
Background
Male HLA-B27-positive radiographic-axial spondyloarthritis (r-axSpA) patients are prone to have severe spinal radiographic progression, but the underlying mechanisms are unclear. ...We recently showed that persistently elevated Lipocalin 2 (LCN2; L) reflects sacroiliac joint (SIJ) inflammation. LCN2 binds to MMP9. Concomitant elevation of L and LCN2-MMP9 (LM) was detected in many inflammatory diseases. We asked whether L and LM play similar roles in r-axSpA pathogenesis.
Methods
We analyzed 190 axSpA patients (123 radiographic and 67 non-radiographic axSpA) who had no detectable circulating Oncostatin M, to avoid complications due to cross-talk between pathways. L and LM levels from a single blood sample of each patient were measured and were correlated with MRI and modified stoke AS (mSASS) scoring. Association of elevated L (L+) or concurrent L+ and elevated LM (LM+) patterns with B27 status and gender were assessed.
Results
In L+LM+ axSpA patients, both L and LM levels correlated with MRI SPARCC SIJ scores, but only LM levels correlated with MRI Berlin Spine Scores, suggesting LM is a biomarker for both SIJ and spinal inflammation. Among patients with minimal spinal ankylosis (mSASSS < 10), 65% of male r-axSpA patients are L+LM+, while 30% and 64% of female patients are L+LM+ and L+, respectively, supporting the role of LM with disease progression. In B27+ L+LM+ male patients, both L and LM (but not CRP) levels correlate with mSASSS. B27 positivity and maleness have additive effects on spondylitis progression, suggesting concurrent high L and LM elevations are associated with B27+ male patients having more significant radiographic damage. L+ B27-negative male patients or L+ female patients are more likely to have milder disease.
Conclusion
L and LM are informative biomarkers for SIJ and spinal inflammation, as well as for ankylosing development in r-axSpA patients. Distinctive L+LM+ or L+ patterns not only could distinguish clinically aggressive vs milder course of disease, respectively, but also provide an explanation for B27-positive male patients being the most susceptible to severe ankylosis.
Informative serum biomarkers for monitoring inflammatory activity and treatment responses in axial spondyloarthritis (axSpA) are lacking. We assessed whether Lipocalin 2 (LCN2) and Oncostatin M ...(OSM), both having roles in inflammation and bone remodeling, may accurately reflect chronic joint inflammation and treatment response in axSpA. Previous reports in animal models showed involvement of LCN2 and OSM in joint/gut inflammation. We asked whether they also play a role in human axSpA.
We analyzed a longitudinal observational axSpA cohort (286 patients) with yearly clinical assessments and concurrent measurements of serum LCN2 and OSM (1204 serum samples) for a mean of 4 years. Biomarker levels were correlated with MRI scoring and treatment response.
Persistent and transient elevation of LCN2 and OSM were observed in axSpA patients. Persistent elevation of LCN2 or OSM, but not CRP, correlated with sacroiliac joint (SIJ) MRI SPARCC scores (Pearson's correlation p = 0.0005 and 0.005 for LCN2 and OSM respectively), suggesting that LCN2/OSM outperforms CRP as reflective of SIJ inflammation. We observed both concordant and discordant patterns of LCN2 and OSM in relationship to back pain, the cardinal clinical symptom in axSpA. Twenty-six percent (73/286) of the patients remained both clinically and serologically active (CASA). Sixty percent (173/286) of the patients became clinically quiescent, with back pain resolved, but 53% (92/173) of them were serologically active (CQSA), indicating that pain control may not indicate control of joint inflammation, as reflected by positive MRI imaging of SIJ. With respect to treatment responses, transient elevation of LCN2 or OSM over time was predictive of better response to all treatments.
In axSpA, persistent LCN2 and/or OSM elevation reflects chronic SIJ inflammation and suboptimal treatment response. In our cohort, half of the currently deemed clinically quiescent patients with back pain resolved continued to demonstrate chronic joint inflammation. LCN2 and OSM profiling outperforms CRP as a predictive measure and provides an objective assessment of chronic local inflammation in axSpA patients.
Little is known about the mechanisms underlying the clinical overlap between gut inflammation and joint ankylosis, as exemplified by the concurrence of inflammatory bowel diseases (IBD) and ...ankylosing spondylitis (AS). As dysbiosis may serve as a common contributor, the anti-microbial pleiotropic factor lipocalin 2 could be a potential mediator due to its roles in inflammation and bone homeostasis.
Baseline colonic pathology was conducted in the ank/ank mouse model. Serum lipocalin 2 was analyzed by ELISA, in ank/ank mutants versus C3FeB6-A/A
wt/wt, in patients with concurrent AS-IBD, AS alone, IBD alone, or mechanical back pain, and in healthy controls. In the ank/ank mouse model, the expression of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) was examined by real-time PCR. Intraperitoneal injection was done with the PPARγ agonist rosiglitazone or antagonist bisphenol A diglycidyl ether for four consecutive days. Serum levels of lipocalin 2 were examined on the sixth day.
This study showed that the ank/ank mice with fully fused spines had concurrent colonic inflammation. By first using the ank/ank mouse model with progressive ankylosis and subclinical colonic inflammation, confirmed in patients with concurrent AS and IBD, elevated circulating lipocalin 2 levels were associated with the coexisting ankylosis and gut inflammation. The intracellular pathway of lipocalin 2 was further investigated with the ank/ank mouse model involving PPARγ. Colonic expression of PPARγ was negatively associated with the degree of gut inflammation. The PPARγ agonist rosiglitazone treatment significantly upregulated the serum levels of lipocalin 2, suggesting a potential regulatory role of PPARγ in the aberrant expression of lipocalin 2.
In summary, lipocalin 2 modulated by PPARγ could be a potential pathway involved in concurrent inflammation and ankylosis in AS and IBD.
Macrophages can display a number of distinct phenotypes, known collectively as polarized macrophages. The best defined of these phenotypes are the classically-activated, interferon gamma (IFNγ)/LPS ...induced (M1) and alternatively-activated, IL-4 induced (M2) macrophages. The goal of this study is to characterize macrophage-Chlamydia interactions in the context of macrophage polarization. Here we use Chlamydia muridarum and murine bone-marrow derived macrophages to show Chlamydia does not induce M2 polarization in macrophages as a survival strategy. Unexpectedly, the infection of macrophages was silent with no upregulation of M1 macrophage-associated genes. We further demonstrate that macrophages polarized prior to infection have a differential capacity to control Chlamydia. M1 macrophages harbor up to 40-fold lower inclusion forming units (IFU) than non-polarized or M2 polarized macrophages. Gene expression analysis showed an increase in 16sRNA in M2 macrophages with no change in M1 macrophages. Suppressed Chlamydia growth in M1 macrophages correlated with the induction of a bacterial gene expression profile typical of persistence as evident by increased Euo expression and decreased Omp1 and Tal expression. Observations of permissive Chlamydia growth in non-polarized and M2 macrophages and persistence in M1 macrophages were supported through electron microscopy. This work supports the importance of IFNγ in the innate immune response to Chlamydia. However, demonstration that the M1 macrophages, despite an antimicrobial signature, fail to eliminate intracellular Chlamydia supports the notion that host-pathogen co-evolution has yielded a pathogen that can evade cellular defenses against this pathogen, and persist for prolonged periods of time in the host.
Objective
To identify an immunologic basis for the male sex bias in ankylosing spondylitis (AS).
Methods
Cohorts of male and female patients with AS and age‐ and sex‐matched healthy control subjects ...were selected, and the levels of serum cytokines (interferon‐γ IFNγ, tumor necrosis factor α, interleukin‐17A IL‐17A, and IL‐6) were examined by enzyme‐linked immunosorbent assay, the frequencies of Th1 and Th17 cells were assessed by flow cytometry, and whole blood gene expression was analyzed using both microarray and NanoString approaches.
Results
The frequency of IL‐17A and Th17 cells, both of which are key factors in the inflammatory Th17 axis, was elevated in male patients with AS but not in female patients with AS. In contrast, AS‐associated alterations in the Th1 axis, such as the frequency of IFNγ and Th1 cells in serum, were independent of a patient's sex. Results of microarray analysis supported an altered Th17 axis in male patients, with a specific increase in IL17RA. In addition, male and female patients with AS displayed shared gene expression patterns, while male patients with AS had additional alterations in gene expression that were not seen in female patients with AS. The differential sex‐related immune profiles were independent of HLA–B27 status, clinical disease activity (as measured by the Bath Ankylosing Spondylitis Disease Activity Index), or treatment (with nonsteroidal antiinflammatory drugs or biologic agents), implicating intrinsic sexual dimorphism in AS.
Conclusion
The results of this study demonstrate distinct sexual dimorphism in the activation status of the immune system in patients with AS, particularly in the Th17 axis. This dimorphism could underlie sex‐related differences in the clinical features of AS and could provide a rationale for sex‐specific treatment of AS.
The mucus layer and gut microbiota interplay contributes to host homeostasis. The mucus layer serves as a scaffold and a carbon source for gut microorganisms; conversely, gut microorganisms, ...including mucin degraders, influence mucin gene expression, glycosylation, and secretion. Conjointly they shield the epithelium from luminal pathogens, antigens, and toxins. Importantly, the mucus layer and gut microbiota are established in parallel during early postnatal life. During this period, the development of gut microbiota and mucus layer is coupled with that of the immune system. Developmental changes of different mucin types can impact the age-dependent patterns of intestinal infection in terms of incidence and severity. Altered mucus layer, dysbiotic microbiota, and abnormal mucus–gut microbiota interaction have the potential for inducing systemic effects, and accompany several intestinal diseases such as inflammatory bowel disease, colorectal cancer, and radiation-induced mucositis. Early life provides a pivotal window of opportunity to favorably modulate the mucus–microbiota interaction. The support of a health-compatible mucin–microbiota maturation in early life is paramount for long-term health and serves as an important opportunity for clinical intervention.
Endoplasmic reticulum-associated aminopeptidase-1 (ERAP1) plays a critical role in the processing of peptides prior to binding to MHC class I molecules. In this article, we show for the first time, ...to our knowledge, that the HLA-B27 immunodominant influenza nucleoprotein (NP) 383-391 epitope is made as an N-terminally extended 14-mer before it is trimmed by ERAP. In the absence of ERAP, there is a significant reduction in the CTL response to the B27/NP383-391 epitope in influenza A (flu)-infected B27/ERAP(-/-) mice. With the use of tetramer staining, the number of naive CD8(+) T cells expressing TCR Vβ8.1 in B27/ERAP(-/-) transgenic mice is significantly lower than that seen in B27/ERAP(+/+) mice. HLA-B27 surface expression in naive and flu-infected B27/ERAP(-/-) mice is also lower than the expression seen for the same allele in naive and flu-infected B27/ERAP(+/+) mice. In contrast, surface expression of HLA-B7 was unaffected by the absence of ERAP in B7/ERAP(-/-) transgenic mice. The B7-restricted NP418-426 CTL response in flu-infected B7/ERAP(-/-) and B7/ERAP(+/+) mice was also similar. These results provide, to our knowledge, the first in vivo demonstration of ERAP functionally influencing host immune response in an HLA allele-specific manner. This principle has relevance to diseases such as ankylosing spondylitis, in which HLA-B27 and ERAP jointly contribute to disease predisposition.
Little is known about the mechanisms underlying the clinical overlap of ankylosing spondylitis (AS) and inflammatory bowel disease (IBD). There is also a lack of reliable serological biomarkers to ...guide AS management. AS is a complex, male-dominant disease with HLA-B27 being the strongest genetic association. The ank/ank mouse is a murine model with serological and pathological features similar to human AS. Lipocalin 2 (murine Lcn2/human LCN2) is a secreted anti-microbial glycoprotein produced in multiple tissues including joint and gut. Given the functions of lipocalin 2 in infection, inflammation and bone remodeling, our aim is to assess whether lipocalin 2 is a key mediator of the gut-joint axis in AS and whether it would serve a better marker for treatment outcomes. The association of LCN2 with HLA-B27 and sex in patients is also analyzed. Chapter 2 shows that aberrant levels of Lcn2 are associated with ankylosis and gut pathology in ank/ank mice, indicating a potential mechanism underlying the overlap of AS and IBD. The interplay of gut inflammation and ankylosis in contributing to elevated Lcn2 levels suggests that the ank/ank mouse is a relevant animal model for the gut-joint interactions, as seen in AS. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a role in aberrant Lcn2 pathway in this AS model. Chapter 3 confirms the elevation of serum LCN2 in AS patients. Levels of LCN2 are associated with coexisting IBD and with the degree of ankylosis. The relationship of HLA-B27 positivity and sex with LCN2 implicates a host-microbe interaction underlying the gut-joint axis in AS. Chapter 4 demonstrates that normalization of LCN2 could be viewed as a treatment target in AS. Serum LCN2 served a more sensitive biomarker for treatment outcome in 89% of AS patients. HLA-B27 status and sex does affect LCN2 as a readout for assessing treatment outcome. The discovery of aberrant Lcn2/LCN2 reveals a relevant mechanism underlying the gut-joint axis in AS. The effect of PPARγ on aberrant Lcn2 pathway sheds lights on novel therapeutic targets for the disease. More knowledge of the dysregulation of Lcn2/LCN2 pathways will contribute to better AS management and more effective treatment strategies.
Protective immunity to the pathogen Chlamydia is dependent on a robust IFN‐γ response generated by innate and adaptive lymphocytes. Here we assess the role of the macrophage in orchestrating a ...protective response in vivo to the murine pathogen, Chlamydia muridarum. During acute pulmonary and peritoneal infection, resident macrophages in both sites are infected with C. muridarum and adopt an inflammatory phenotype. In the lung, this activation is restricted to interstitial macrophages, which harbor higher levels of C. muridarum 16sRNA than alveolar macrophages. We examined innate and adaptive lymphocyte activation in the peritoneal cavity with macrophage depletion and with adoptive transfer of infected macrophages. These experiments demonstrate macrophage activation correlates with a protective IFN‐γ response and effective control of C. muridarum. These studies suggest that a quantitative or qualitative alteration in macrophages may play a key role in the development of Chlamydia‐associated diseases.
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