Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Phage Therapy for Respiratory Infections: Opportunities and Challenges.

We are entering the post-antibiotic era. Antimicrobial resistance (AMR) is a critical problem in chronic lung infections resulting in progressive respiratory failure and increased mortality. In the absence of emerging novel antibiotics to counter AMR infections, bacteriophages (phages), viruses that infect bacteria, have become a promising option for chronic respiratory infections. However, while personalized phage therapy is associated with improved outcomes in individual cases, clinical trials demonstrating treatment efficacy are lacking, limiting the therapeutic potential of this approach for respiratory infections. In this review, we address the current state of phage therapy for managing chronic respiratory diseases. We then discuss how phage therapy may address major microbiologic obstacles which hinder disease resolution of chronic lung infections with current antibiotic-based treatment practices. Finally, we highlight the challenges that must be addressed for successful phage therapy clinical trials. Through this discussion, we hope to expand on the potential of phages as an adjuvant therapy in chronic lung infections, as well as the microbiologic challenges that need to be addressed for phage therapy to expand beyond personalized salvage therapy.

IL-10 promotes endothelial progenitor cell infiltration and wound healing via STAT3.

Endothelial progenitor cells (EPCs) contribute to de novo angiogenesis, tissue regeneration, and remodeling. Interleukin 10 (IL-10), an anti-inflammatory cytokine that primarily signals via STAT3, has been shown to drive EPC recruitment to injured tissues. Our previous work demonstrated that overexpression of IL-10 in dermal wounds promotes regenerative tissue repair via STAT3-dependent regulation of fibroblast-specific hyaluronan synthesis. However, IL-10's role and specific mode of action on EPC recruitment, particularly in dermal wound healing and neovascularization in both normal and diabetic wounds, remain to be defined. Therefore, inducible skin-specific STAT3 knockdown mice were studied to determine IL-10's impact on EPCs, dermal wound neovascularization and healing, and whether it is STAT3-dependent. We show that IL-10 overexpression significantly elevated EPC counts in the granulating wound bed, which was associated with robust capillary lumen density and enhanced re-epithelialization of both control and diabetic (db/db) wounds at day 7. We noted increased VEGF and high C-X-C motif chemokine 12 (CXCL12) levels in wounds and a favorable CXCL12 gradient at day 3 that may support EPC mobilization and infiltration from bone marrow to wounds, an effect that was abrogated in STAT3 knockdown wounds. These findings were supported in vitro. IL-10 promoted VEGF and CXCL12 synthesis in primary murine dermal fibroblasts, with blunted VEGF expression upon blocking CXCL12 in the media by antibody binding. IL-10-conditioned fibroblast media also significantly promoted endothelial sprouting and network formation. In conclusion, these studies demonstrate that overexpression of IL-10 in dermal wounds recruits EPCs and leads to increased vascular structures and faster re-epithelialization.

Plasma Microbial Cell-free DNA Next-generation Sequencing in the Diagnosis and Management of Febrile Neutropenia.

BACKGROUND: Standard testing fails to identify a pathogen in most patients with febrile neutropenia (FN). We evaluated the ability of the Karius microbial cell-free DNA sequencing test (KT) to identify infectious etiologies of FN and its impact on antimicrobial management. METHODS: This prospective study (ClinicalTrials.gov; NCT02912117) enrolled and analyzed 55 patients with FN. Up to 5 blood samples were collected per subject within 24 hours of fever onset (T1) and every 2 to 3 days. KT results were compared with blood culture (BC) and standard microbiological testing (SMT) results. RESULTS: Positive agreement was defined as KT identification of ≥1 isolate also detected by BC. At T1, positive and negative agreement were 90% (9/10) and 31% (14/45), respectively; 61% of KT detections were polymicrobial. Clinical adjudication by 3 independent infectious diseases specialists categorized Karius results as: unlikely to cause FN (N = 0); definite (N = 12): KT identified ≥1 organism also found by SMT within 7 days; probable (N = 19): KT result was compatible with a clinical diagnosis; possible (N = 10): KT result was consistent with infection but not considered a common cause of FN. Definite, probable, and possible cases were deemed true positives. Following adjudication, KT sensitivity and specificity were 85% (41/48) and 100% (14/14), respectively. Calculated time to diagnosis was generally shorter with KT (87%). Adjudicators determined real-time KT results could have allowed early optimization of antimicrobials in 47% of patients, by addition of antibacterials (20%) (mostly against anaerobes [12.7%]), antivirals (14.5%), and/or antifungals (3.6%); and antimicrobial narrowing in 27.3% of cases. CLINICAL TRIALS REGISTRATION: NCT02912117. CONCLUSION: KT shows promise in the diagnosis and treatment optimization of FN.

Chronic suppurative otitis media causes macrophage-associated sensorineural hearing loss.

BACKGROUND: Chronic suppurative otitis media (CSOM) is the most common cause of permanent hearing loss in children in the developing world. A large component of the permanent hearing loss is sensory in nature and our understanding of the mechanism of this has so far been limited to post-mortem human specimens or acute infection models that are not representative of human CSOM. In this report, we assess cochlear injury in a validated Pseudomonas aeruginosa (PA) CSOM mouse model. METHODS: We generated persisters (PCs) and inoculated them into the mouse middle ear cavity. We tracked infection with IVIS and detected PA using RT-PCR. We assessed cochlear damage and innate immunity by Immunohistochemistry. Finally, we evaluated cytokines with multiplex assay and quantitative real-time PCR. RESULTS: We observed outer hair cell (OHC) loss predominantly in the basal turn of the cochlear at 14 days after bacterial inoculation. Macrophages, not neutrophils are the major immune cells in the cochlea in CSOM displaying increased numbers and a distribution correlated with the observed cochlear injury. The progression of the morphological changes suggests a transition from monocytes into tissue macrophages following infection. We also show that PA do not enter the cochlea and live bacteria are required for cochlear injury. We characterized cytokine activity in the CSOM cochlea. CONCLUSIONS: Taken together, this data shows a critical role for macrophages in CSOM-mediated sensorineural hearing loss (SNHL).

Weekly injection of IL-2 using an injectable hydrogel reduces autoimmune diabetes incidence in NOD mice.

AIMS/HYPOTHESIS: IL-2 injections are a promising therapy for autoimmune type 1 diabetes but the short half-life of this cytokine in vivo limits effective tissue exposure and necessitates frequent injections. Here we have investigated whether an injectable hydrogel could be used to promote prolonged IL-2 release in vivo. METHODS: Capitalising on the IL-2-binding capabilities of heparin, an injectable hydrogel incorporating clinical-grade heparin, collagen and hyaluronan polymers was used to deliver IL-2. The IL-2-release kinetics and in vivo stability of this material were examined. The ability of soluble IL-2 vs hydrogel-mediated IL-2 injections to prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes were compared. RESULTS: We observed in vitro that the hydrogel released IL-2 over a 12-day time frame and that injected hydrogel likewise persisted 12 days in vivo. Notably, heparin binding potentiates the activity of IL-2 and enhances IL-2- and TGFß-mediated expansion of forkhead box P3-positive regulatory T cells (FOXP3+ Tregs). Finally, weekly administration of IL-2-containing hydrogel partially prevented autoimmune diabetes while injections of soluble IL-2 did not. CONCLUSIONS/INTERPRETATION: Hydrogel delivery may reduce the number of injections required in IL-2 treatment protocols for autoimmune diabetes. Graphical abstract.

Phage Therapy for Limb-threatening Prosthetic Knee Klebsiella pneumoniae Infection: Case Report and In Vitro Characterization of Anti-biofilm Activity.

BACKGROUND: Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention. METHODS: The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient's isolate and determined by safranin staining. RESULTS: Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections.

Interleukin-10 Producing T Lymphocytes Attenuate Dermal Scarring.

OBJECTIVE: Demonstrate the impact of IL-10 producing T lymphocytes on mediating dermal scarring. SUMMARY BACKGROUND DATA: We demonstrated that CD4+ cells are essential to improving postinjury wound healing and preventing fibrosis. CD4+ subsets secrete differential cytokine and growth factor profiles, though their role in fibrosis is not known. IL-10, a key anti-inflammatory cytokine shown to promote regenerative wound healing, is secreted by some CD4+ subsets. We, therefore, hypothesize that IL-10 producing CD4+ T lymphocyte subsets selectively attenuate dermal wound fibrosis. METHODS: IL-10-/- and wild-type murine splenocytes were enriched for CD4+ lymphocytes and adoptively transferred into severe combined immunodeficient (SCID) mice that received full-thickness wounds which were analyzed at days 7 and 28 for inflammation and collagen content. We then sorted CD4+CD44int/lowFoxP3-CD62L+ T cells (Tnaive) or CD4+CD44HiFoxP3- type 1 regulatory (Tr1) T cell subsets from 10BiT murine splenocytes, activated them, and transferred them into wounds. In vitro, dermal fibroblasts were cocultured with Tnaive or Tr1 and the effect on extracellular matrix (ECM) regulation was analyzed. RESULTS: The anti-inflammatory and antifibrotic effects of CD4+ cells on SCID wounds were lost with cells from IL-10-/- mice. Adoptive transfer of Tr1 into SCID mice resulted in accelerated wound closure at d7 with reduced fibrosis at d28, with Tr1 favoring hyaluronan production by fibroblasts, an ECM molecule implicated in IL-10-induced regenerative healing. CONCLUSIONS: IL-10 producing T-lymphocytes, specifically Tr1, regulate inflammatory cell cytokine expression to promote HA-rich ECM deposition and attenuate fibrosis. Promoting IL-10 producing lymphocytes in wounds may be a therapeutic target to promote regenerative wound healing.

Dynamic light scattering microrheology for soft and living materials.

We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. This microrheology technique only requires a small sample volume of 12 µL to measure up to six decades in time of rheological behavior. We demonstrate the use of dynamic light scattering microrheology (DLSµR) on a variety of soft materials, including dilute polymer solutions, covalently-crosslinked polymer gels, and active, biological fluids. In this work, we detail the procedure for applying the technique to new materials and discuss the critical considerations for implementing the technique, including a custom analysis script for analyzing data output. We focus on the advantages of applying DLSµR to biologically relevant materials: breast cancer cells encapsulated in a collagen gel and cystic fibrosis sputum. DLSµR is an easy, efficient, and economical rheological technique that can guide the design of new polymeric materials and facilitate the understanding of the underlying physics governing behavior of naturally derived materials.

Tolerant Small-colony Variants Form Prior to Resistance Within a Staphylococcus aureus Biofilm Based on Antibiotic Selective Pressure.

BACKGROUND: The treatment of periprosthetic joint infection (PJI) is focused on the surgical or chemical removal of biofilm. Antibiotics in isolation are typically ineffective against PJI. Bacteria survive after antibiotic administration because of antibiotic tolerance, resistance, and persistence that arise in the resident bacteria of a biofilm. Small-colony variants are typically slow-growing bacterial subpopulations that arise after antibiotic exposure and are associated with persistent and chronic infections such as PJI. The role of biofilm-mediated antibiotic tolerance in the emergence of antibiotic resistance remains poorly defined experimentally. QUESTIONS/PURPOSES: We asked: (1) Does prior antibiotic exposure affect how Staphylococcus aureus survives within a developing biofilm when exposed to an antibiotic that penetrates biofilm, like rifampicin? (2) Does exposure to an antibiotic with poor biofilm penetration, such as vancomycin, affect how S. aureus survives within a developing biofilm? (3) Do small-colony variants emerge from antibiotic-tolerant or -resistant bacteria in a S. aureus biofilm? METHODS: We used a porous membrane as an in vitro implant model to grow luminescent S. aureus biofilms and simultaneously track microcolony expansion. We evaluated the impact of tolerance on the development of resistance by comparing rifampicin (an antibiotic that penetrates S. aureus biofilm) with vancomycin (an antibiotic that penetrates biofilm poorly). We performed viability counting after membrane dissociation to discriminate among tolerant, resistant, and persistent bacteria. Biofilm quantification and small-colony morphologies were confirmed using scanning electron microscopy. Because of experimental variability induced by the starting bacterial inoculum, relative changes were compared since absolute values may not have been statistically comparable. RESULTS: Antibiotic-naïve S. aureus placed under the selective pressure of rifampicin initially survived within an emerging biofilm by using tolerance given that biofilm resident cell viability revealed 1.0 x 108 CFU, of which 7.5 x 106 CFU were attributed to the emergence of resistance and 9.3 x 107 CFU of which were attributed to the development of tolerance. Previous exposure of S. aureus to rifampicin obviated tolerance-mediate survival when rifampicin resistance was present, since the number of viable biofilm resident cells (9.5 x 109 CFU) nearly equaled the number of rifampicin-resistant bacteria (1.1 x 1010 CFU). Bacteria exposed to an antibiotic with poor biofilm penetration, like vancomycin, survive within an emerging biofilm by using tolerance as well because the biofilm resident cell viability for vancomycin-naïve (1.6 x 1010 CFU) and vancomycin-resistant (1.0 x 1010 CFU) S. aureus could not be accounted for by emergence of resistance. Adding rifampicin to vancomycin resulted in a nearly 500-fold reduction in vancomycin-tolerant bacteria from 1.5 x 1010 CFU to 3.3 x 107 CFU. Small-colony variant S. aureus emerged within the tolerant bacterial population within 24 hours of biofilm-penetrating antibiotic administration. Scanning electron microscopy before membrane dissociation confirmed the presence of small, uniform cells with biofilm-related microstructures when unexposed to rifampicin as well as large, misshapen, lysed cells with a small-colony variant morphology [29, 41, 42, 63] and a lack of biofilm-related microstructures when exposed to rifampicin. This visually confirmed the rapid emergence of small-colony variants within the sessile niche of a developing biofilm when exposed to an antibiotic that exerted selective pressure. CONCLUSION: Tolerance explains why surgical and nonsurgical modalities that rely on antibiotics to "treat" residual microscopic biofilm may fail over time. The differential emergence of resistance based on biofilm penetration may explain why some suppressive antibiotic therapies that do not penetrate biofilm well may rely on bacterial control while limiting the emergence of resistance. However, this strategy fails to address the tolerant bacterial niche that harbors persistent bacteria with a small-colony variant morphology. CLINICAL RELEVANCE: Our work establishes biofilm-mediated antibiotic tolerance as a neglected feature of bacterial communities that prevents the effective treatment of PJI.

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition.

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

Phosphorylation of αB-crystallin supports reactive astrogliosis in demyelination.

The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.

Hyaluronan content governs tissue stiffness in pancreatic islet inflammation.

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Salivary Thiocyanate as a Biomarker of Cystic Fibrosis Transmembrane Regulator Function.

Improved methods are needed to reliably assess Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in vivo in light of recent therapeutic developments targeting the CFTR protein. Oral fluid from patients with cystic fibrosis (CF) and healthy controls (HCs) were studied using colorimetry and nonresonant Raman spectroscopy. Colorimetry experiments showed only a 36% decrease in thiocyanate (SCN-) concentration, but a sharp Raman peak at 2068 cm-1, attributable to (SCN-) vibrations, normalized to C-H peak, was on average 18 times higher for HC samples. Samples from patients undergoing treatment with CFTR modulators including ivacaftor, lumacaftor, and tezacaftor showed a high normalized peak in response to therapy. The peak intensity was consistent in longitudinal samples from single donors and in stored samples. The Raman peak ratio is a more sensitive, convenient, noninvasive biomarker for assessments of the therapeutic efficacy of drugs targeting CFTR and provides a value that is in much better agreement with theoretical expectations of saliva SCN- concentrations compared to colorimetry. This insight may greatly facilitate assessments of CFTR modulator efficacy in individual patients.

A Consensus Definitive Classification of Scavenger Receptors and Their Roles in Health and Disease.

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.

The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.

The cytokine IL-10 has potent antifibrotic effects in models of adult fibrosis, but the mechanisms of action are unclear. Here, we report a novel finding that IL-10 triggers a signal transducer and activator of transcription 3 (STAT3)-dependent signaling pathway that regulates hyaluronan (HA) metabolism and drives adult fibroblasts to synthesize an HA-rich pericellular matrix, which mimics the fetal regenerative wound healing phenotype with reduced fibrosis. By using cre-lox-mediated novel, inducible, fibroblast-, keratinocyte-, and wound-specific STAT3-knockdown postnatal mice-plus syngeneic fibroblast cell-transplant models-we demonstrate that the regenerative effects of IL-10 in postnatal wounds are dependent on HA synthesis and fibroblast-specific STAT3-dependent signaling. The importance of IL-10-induced HA synthesis for regenerative wound healing is demonstrated by inhibition of HA synthesis in a murine wound model by administering 4-methylumbelliferone. Although IL-10 and STAT3 signaling were intact, the antifibrotic repair phenotype that is induced by IL-10 overexpression was abrogated in this model. Our data show a novel role for IL-10 beyond its accepted immune-regulatory mechanism. The opportunity for IL-10 to regulate a fibroblast-specific formation of a regenerative, HA-rich wound extracellular matrix may lead to the development of innovative therapies to attenuate postnatal fibrosis in organ systems or diseases in which dysregulated inflammation and HA intersect.-Balaji, S., Wang, X., King, A., Le, L. D., Bhattacharya, S. S., Moles, C. M., Butte, M. J., de Jesus Perez, V. A., Liechty, K. W., Wight, T. N., Crombleholme, T. M., Bollyky, P. L., Keswani, S. G. Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.

Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.