Brian T. Campfield

Follistatin‐like protein 1 enhances NLRP3 inflammasome‐mediated IL‐1β secretion from monocytes and macrophages

Follistatin‐like protein 1 (FSTL‐1) is overexpressed in a number of inflammatory conditions characterized by elevated IL‐1β. Here, we found that FSTL‐1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL‐1 to IL‐1β secretion, WT and FSTL‐1‐deficient mice were injected with LPS. While LPS induced IL‐1β in the sera of WT mice, it was low or undetectable in FSTL‐1‐deficient mice. Monocytes/macrophages, a key source of IL‐1β, do not normally express FSTL‐1. However, FSTL‐1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL‐1 at sites of inflammation. In vitro, intracellular FSTL‐1 localized to the mitochondria. FSTL‐1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL‐1β secretion. FSTL‐1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus‐mediated overexpression of FSTL‐1 in mouse paws led to activation of the inflammasome complex and local secretion of IL‐1β and IL‐1β‐related proinflammatory cytokines. These results suggest that FSTL‐1 may act on the NLRP3 inflammasome to promote IL‐1β secretion from monocytes/macrophages.

Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells

Objectives Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. Methods To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. Results The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor β signalling in FSTL1 KO cells was significantly suppressed. Conclusions FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.

Eosinophils Contribute to Early Clearance of Pneumocystis murina Infection

Pneumocystis pneumonia remains a common opportunistic infection in the diverse immunosuppressed population. One clear risk factor for susceptibility to Pneumocystis is a declining CD4+ T cell count in the setting of HIV/AIDS or primary immunodeficiency. Non–HIV-infected individuals taking immunosuppressive drug regimens targeting T cell activation are also susceptible. Given the crucial role of CD4+ T cells in host defense against Pneumocystis, we used RNA sequencing of whole lung early in infection in wild-type and CD4-depleted animals as an unbiased approach to examine mechanisms of fungal clearance. In wild-type mice, a strong eosinophil signature was observed at day 14 post Pneumocystis challenge, and eosinophils were increased in the bronchoalveolar lavage fluid of wild-type mice. Furthermore, eosinophilopoiesis-deficient Gata1tm6Sho/J mice were more susceptible to Pneumocystis infection when compared with BALB/c controls, and bone marrow–derived eosinophils had in vitro Pneumocystis killing activity. To drive eosinophilia in vivo, Rag1−/− mice were treated with a plasmid expressing IL-5 (pIL5) or an empty plasmid control via hydrodynamic injection. The pIL5-treated mice had increased serum IL-5 and eosinophilia in the lung, as well as reduced Pneumocystis burden, compared with mice treated with control plasmid. In addition, pIL5 treatment could induce eosinophilia and reduce Pneumocystis burden in CD4-depleted C57BL/6 and BALB/c mice, but not eosinophilopoiesis-deficient Gata1tm6Sho/J mice. Taken together, these results demonstrate that an early role of CD4+ T cells is to recruit eosinophils to the lung and that eosinophils are a novel candidate for future therapeutic development in the treatment of Pneumocystis pneumonia in the immunosuppressed population.

Follistatin-like protein 1 is a critical mediator of experimental Lyme arthritis and the humoral response to Borrelia burgdorferi infection

Follistatin-like protein 1 (FSTL-1) has recently been described as a critical mediator of CIA and a marker of disease activity. Lyme arthritis, caused by Borrelia burgdorferi, shares similarities with autoimmune arthritis and the experimental murine model collagen-induced arthritis (CIA). Because FSTL-1 is important in CIA and autoimmune arthritides, and Lyme arthritis shares similarities with CIA, we hypothesized that FSTL-1 may be an important mediator of Lyme arthritis. We demonstrate for the first time that FSTL-1 is induced by B. burgdorferi infection and is required for the development of Lyme arthritis in a murine model, utilizing a gene insertion to generate FSTL-1 hypomorphic mice. Using qPCR and qRT-PCR, we found that despite similar early infectious burden, FSTL-1 hypomorphic mice have improved spirochetal clearance in the face of attenuated arthritis and inflammatory cytokine production. Further, FSTL-1 mediates pathogen-specific antibody production and antigen recognition when assessed by ELISA and one- and two-dimensional immunoblotting. This study is the first to describe a role for FSTL-1 in the development of Lyme arthritis and anti-Borrelia response, and the first to demonstrate a role for FSTL-1 in response to infection, highlighting the potential for FSTL-1 as a target in the treatment of B. burgdorferi infection.

The DBA/1 strain is a novel mouse model for experimental Borrelia burgdorferi infection.

ABSTRACT Lyme arthritis, caused by Borrelia burgdorferi, has similarities to rheumatoid arthritis and its experimental murine model, collagen-induced arthritis (CIA). Currently, no common strain exists for examination of arthritis models of Lyme arthritis and CIA, which are typically studied in C3H/HeJ and DBA/1 mice, respectively. The aim of this study was to define the characteristics of Borrelia burgdorferi infection and arthritis in the DBA/1 murine strain. Murine Lyme arthritis was induced in C3H/HeJ and DBA/1 mice by subcutaneous infection with B. burgdorferi. Tibiotarsal joints were measured during infection, and mice were sacrificed for histologic, microbiologic, and serologic analysis on days 14 and 42 postinfection. All bladder cultures obtained from C3H/HeJ and DBA/1 mice at 14 days postinfection grew Borrelia. There was no significant difference in spirochetal burdens in hearts and tibiotarsal joints at days 14 and 42 postinfection. Tibiotarsal joint swelling and histologic scoring were not significantly different between the two strains. Serologic analysis revealed increased IgG2a production in C3H/HeJ mice compared to DBA/1 mice. Analysis of 2-dimensional immunoblots revealed several specific antigens (LA7, BBA03, BBA64, BBA73, OspA, and VlsE) which were not recognized by DBA/1 sera. We conclude that the DBA/1 murine strain is a suitable model for the study of Lyme arthritis and experimental B. burgdorferi infection, allowing direct comparison between Lyme arthritis and collagen-induced arthritis. The specificity of the humoral immune response differs between the two strains, further study of which may reveal important findings about how individual strains respond to B. burgdorferi infection.

Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

Follistatin‐like protein 1 (FSTL‐1) possesses several newly identified roles in mammalian biology, including interleukin (IL)‐17‐driven inflammation, though the mechanism underlying FSTL‐1 influence on IL‐17‐mediated cytokine production is unknown. Using parallel in vitro bone marrow stromal cell models of FSTL‐1 suppression, we employed unbiased microarray analysis to identify FSTL‐1‐regulated genes and pathways that could influence IL‐17‐dependent production of IL‐6 and granulocyte colony‐stimulating factor. We discovered that FSTL‐1 modulates Il17rc gene expression. Specifically, FSTL‐1 was necessary for Il17rc gene transcription, IL‐17RC surface protein expression and IL‐17‐dependent cytokine production. This work identifies a mechanism by which FSTL‐1 influences IL‐17‐driven inflammatory signaling in vitro and reveals a novel function for FSTL‐1, as a modulator of gene expression. Thus enhanced understanding of the interplay between FSTL‐1 and IL‐17‐mediated inflammation may provide insight into potential therapeutic targets of IL‐17‐mediated diseases and warrants ongoing study of in vivo models and clinical scenarios of FSTL‐1‐influenced diseases.

Oral Management for Pediatric Lyme Meningitis

Background Guidelines for pediatric Lyme meningitis recommend treatment with parenteral therapy [1, 2]. Adult studies suggest that Lyme meningitis can be successfully treated with oral therapy. Our objective was to evaluate the clinical response, side effects and outcome of oral therapy for Lyme meningitis in the pediatric population compared with parenteral therapy in an area endemic for Lyme disease. Methods We conducted a case series chart review from January 2012 to May 2017 of pediatrics patient diagnosed and treated for Lyme meningitis. We recorded clinical presentation, laboratory values, antimicrobial therapy and follow up after therapy to compare the efficacy of oral versus parenteral route of therapy. Results We identified 38 patients diagnosed with Lyme meningitis. Thirty-two patients were discharge with exclusively oral therapy with: doxycycline and amoxicillin. We had only 2 patients developed potential adverse effects from oral doxycycline therapy. All patients treated with oral antibiotics had resolution of symptoms on follow up appointments. Conclusions Oral therapy for Lyme meningitis yields no serious adverse events, was well tolerated and showed resolution of symptoms.

Insights From the Geographic Spread of the Lyme Disease Epidemic

Background Lyme disease is the most common reportable zoonotic infection in the United States. Recent data suggest spread of the Ixodes tick vector and increasing incidence of Lyme disease in several states, including Pennsylvania. We sought to determine the clinical presentation and healthcare use patterns for pediatric Lyme disease in western Pennsylvania. Methods The electronic medical records of all patients with an International Classification of Disease, Ninth Revision, diagnosis of Lyme disease between 2003 and 2013 at Childrens Hospital of Pittsburgh were individually reviewed to identify confirmed cases of Lyme disease. The records of 773 patients meeting these criteria were retrospectively analyzed for patient demographics, disease manifestations, and healthcare use. Results An Lyme disease increased exponentially in the pediatric population of western Pennsylvania. There was a southwestward migration of Lyme disease cases, with a shift from rural to nonrural zip codes. Healthcare provider involvement evolved from subspecialists to primary care pediatricians and emergency departments (EDs). Patients from nonrural zip codes more commonly presented to the ED, while patients from rural zip codes used primary care pediatricians and EDs equally. Conclusions The current study details the conversion of western Pennsylvania from a Lyme-naive to a Lyme-epidemic area, highlighting changes in clinical presentation and healthcare use over time. Presenting symptoms and provider type differed between those from rural and nonrural zip codes. By elucidating the temporospatial epidemiology and healthcare use for pediatric Lyme disease, the current study may inform public health measures regionally while serving as an archetype for other areas at-risk for Lyme disease epidemics.