In vivo single-cell high-dimensional mass cytometry analysis to track the interactions between Klebsiella pneumoniae and myeloid cells.

In vivo single-cell approaches have transformed our understanding of the immune populations in tissues. Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interactions with bacteria, a method we term Bac-CyTOF. We focus on the pathogen Klebsiella pneumoniae interrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with a K. pneumoniae hypervirulent strain. The atlas is characterized by a decrease in the populations of alveolar and monocyte-derived macrophages. Conversely, neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers of less active cells such as the immune checkpoint PD-L1. These are the cells infected. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shifting Klebsiella from alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. The lack of T6SS results in an increase of cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By probing Klebsiella, and Acinetobacter baumannii strains with limited ability to survive in vivo, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of mice clearing infections. We leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING in Klebsiella infections. sting-/- infected mice present features consistent with clearing the infection including the reduced levels of PD-L1. STING absence facilitates Klebsiella clearance.

In vivo single-cell transcriptomics reveal Klebsiella pneumoniae skews lung macrophages to promote infection.

The strategies deployed by antibiotic-resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host-pathogen interaction resulting in skewing lung macrophage polarisation by the human pathogen Klebsiella pneumoniae. We identify interstitial macrophages (IMs) as the main population of lung macrophages associated with Klebsiella. Single-cell transcriptomics and trajectory analysis of cells reveal type I IFN and IL10 signalling, and macrophage polarisation are characteristic of infected IMs, whereas Toll-like receptor (TLR) and Nod-like receptor signalling are features of infected alveolar macrophages. Klebsiella-induced macrophage polarisation is a singular M2-type we termed M(Kp). To rewire macrophages, Klebsiella hijacks a TLR-type I IFN-IL10-STAT6 axis. Absence of STAT6 limits Klebsiella intracellular survival and facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. Capsule polysaccharide governs M(Kp). Klebsiella also skews human macrophage polarisation towards M(Kp) in a type I IFN-IL10-STAT6-dependent manner. Klebsiella induction of M(Kp) represents a novel strategy to overcome host restriction, and identifies STAT6 as target to boost defences against Klebsiella.

Deletion of Socs3 in LysM+ cells and Cx3cr1 resulted in age-dependent development of retinal microgliopathy.

BACKGROUND: We generated a mouse model of primary microglial dysfunction by deleting two negative immune regulatory genes, Cx3cr1 and Socs3 (in LysM+ cells). This study aimed to understand how primary microglial dysfunction impacts retinal neurons during aging. METHODS: The LysMCre-Socs3fl/flCx3cr1gfp/gfp double knockout (DKO), LysMCre-Socs3fl/fl, Cx3cr1gfp/gfp and Socs3fl/fl mice were maintained up to 12 months. Eyes were collected and processed for immunohistochemistry of IBA-1, cone arrestin, secretagogin, PKCα and GABA. Brain microglia from DKO and WT mice were stimulated with LPS + IFN-γ or IL-4. The expression of TNF-α, IL-1ß, IL-6, iNOS, IL-12p40, IL-23p19, CCL2, CCL5, CXCL2, IL-10, CD206 and Arg1 were examined by qRT-PCR and protein production was measured by Luminex assay. Retinal explants from C57BL/6 J mice were co-cultured with microglia from DKO or WT mice for 24 h, after which the number of cone arrestin+ cells in retinal flatmount were quantified. RESULTS: In 3-5 month old mice, the number of microglia in retinal ganglion cell layer (GCL) and inner plexiform layer (IPL) were comparable in all strains of mice. The DKO mice had a significantly higher number of microglia in the outer plexiform layer (OPL) but significantly lower numbers of cone arrestin+, secretagogin+ and GABA+ cells compared to Socs3fl/fl and single KO mice. During aging, 57% of the DKO mice died before 12 months old. The 10-12 months old DKO mice had significantly higher numbers of microglia in GCL/IPL and OPL than age-matched Socs3fl/fl and single KO mice. The aged DKO mice developed retinal pigment epithelial (RPE) dysmorphology accompanied by subretinal microglial accumulation. The number of photoreceptors, bipolar cells (Secretagogin+ or PKCα+) and GABA+ amacrine cells was significantly lower in aged DKO mice compared to age-matched Socs3fl/fl and single KO mice. Microglia from DKO mice showed significantly higher levels of phagocytic activity and produced higher levels of TNF-α, IL-6, CCL2, CCL5, CXCL2 and CXCL10 compared to microglia from Socs3fl/fl mice. Co-culture of retinal explants with LPS + IFN-γ or IL-4 pre-treated DKO microglia significantly reduced cone photoreceptor survival. CONCLUSIONS: The LysMCre-Socs3fl/flCx3cr1gfp/gfp DKO mice displayed primary microglial dysfunction and developed age-related retinal microgliopathy characterized by aggragated microglial activation and multiple retinal neuronal and RPE degeneration. TRIAL REGISTRATION: Not applicable. The article does not contain any results from human participants.

Hyaloid Vasculature as a Major Source of STAT3+ (Signal Transducer and Activator of Transcription 3) Myeloid Cells for Pathogenic Retinal Neovascularization in Oxygen-Induced Retinopathy.

OBJECTIVE: Myeloid cells are critically involved in inflammation-induced angiogenesis, although their pathogenic role in the ischemic retina remains controversial. We hypothesize that myeloid cells contribute to pathogenic neovascularization in retinopathy of prematurity through STAT3 (signal transducer and activator of transcription 3) activation. Approach and Results: Using the mouse model of oxygen-induced retinopathy, we show that myeloid cells (CD45+IsolectinB4 [IB4]+) and particularly M2-type macrophages (CD45+ Arg1+), comprise a major source of STAT3 activation (pSTAT3) in the immature ischemic retina. Most of the pSTAT3-expressing myeloid cells concentrated at the hyaloid vasculature and their numbers were strongly correlated with the severity of pathogenic neovascular tuft formation. Pharmacological inhibition of STAT3 reduced the load of IB4+ cells in the hyaloid vasculature and significantly reduced the formation of pathogenic neovascular tufts during oxygen-induced retinopathy, leading to improved long-term visual outcomes (ie, increased retinal thickness and scotopic b-wave electroretinogram responses). Genetic deletion of SOCS3 (suppressor of cytokine signaling 3), an endogenous inhibitor of STAT3, in myeloid cells, enhanced pathological and physiological neovascularization in oxygen-induced retinopathy, indicating that myeloid-STAT3 signaling is crucial for retinal angiogenesis. CONCLUSIONS: Circulating myeloid cells may migrate to the immature ischemic retina through the hyaloid vasculature and contribute to retinal neovascularization via activation of STAT3. Understanding how STAT3 modulates myeloid cells for vascular repair/pathology may provide novel therapeutic options in pathogenic angiogenesis.

FKBPL-based peptide, ALM201, targets angiogenesis and cancer stem cells in ovarian cancer.

BACKGROUND: ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. METHODS: In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). RESULTS: ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. CONCLUSION: FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

STAT3 activation in circulating myeloid-derived cells contributes to retinal microvascular dysfunction in diabetes.

BACKGROUND: Leukostasis is a key patho-physiological event responsible for capillary occlusion in diabetic retinopathy. Circulating monocytes are the main cell type entrapped in retinal vessels in diabetes. In this study, we investigated the role of the signal transducer and activator of transcription 3 (STAT3) pathway in diabetes-induced immune cell activation and its contribution to retinal microvascular degeneration. METHODS: Forty-one patients with type 1 diabetes (T1D) [mild non-proliferative diabetic retinopathy (mNPDR) (n = 13), active proliferative DR (aPDR) (n = 14), inactive PDR (iPDR) (n = 14)] and 13 age- and gender-matched healthy controls were recruited to the study. C57BL/6 J WT mice, SOCS3fl/fl and LysMCre/+SOCS3fl/fl mice were rendered diabetic by Streptozotocin injection. The expression of the phosphorylated human and mouse STAT3 (pSTAT3), mouse LFA-1, CD62L, CD11b and MHC-II in circulating immune cells was evaluated by flow cytometry. The expression of suppressor of cytokine signalling 3 (SOCS3) was examined by real-time RT-PCR. Mouse plasma levels of cytokines were measured by Cytometric Beads Array assay. Retinal leukostasis was examined following FITC-Concanavalin A perfusion and acellular capillary was examined following Isolectin B4 and Collagen IV staining. RESULTS: Compared to healthy controls, the expression of pSTAT3 in circulating leukocytes was statistically significantly higher in mNPDR but not aPDR and was negatively correlated with diabetes duration. The expression of pSTAT3 and its inhibitor SOCS3 was also significantly increased in leukocytes from diabetic mice. Diabetic mice had higher plasma levels of IL6 and CCL2 compared with control mice. LysMCre/+SOCS3fl/fl mice and SOCS3fl/fl mice developed comparative levels of diabetes, but leukocyte activation, retinal leukostasis and number of acellular capillaries were statistically significantly increased in LysMCre/+SOCS3fl/fl diabetic mice. CONCLUSION: STAT3 activation in circulating immune cells appears to contribute to retinal microvascular degeneration and may be involved in DR initiation in T1D.

Development of transplant immunosuppressive agents - considerations in the use of animal models.

INTRODUCTION: The development of all immunosuppressant agents to date has involved the experimental use of large and small animal models. Over the last half-century, immunosuppressive drugs have extended the lives of transplant patients worldwide. However, the use of animal models in the development of these drugs is not perfect, and this has brought to light a number of issues including idiosyncratic reactions that are found in animal models but not in humans. The 2006 highly publicized case of the 'elephant man' TGN 1412 drug trial highlights the importance of being cogent of the limitations of animal models. Areas covered: This review covers the utility and limitations of the use of animal models for the development of immunosuppressant agents. This includes both large and small animal models, particularly rodent models in the transplant setting. Expert opinion: The use of animal models represents a critical stage in the development of immunosuppressive drugs. Limitations include physiological differences to humans; this is especially true of immunologically naïve lab rodents with small memory cell populations. Toxic drug levels may differ widely between species. Animal models are also costly and raise ethical concerns. However, there is currently no way to recreate the complex environment of the human immune system purely in vitro.

Microneedle arrays for vaccine delivery: the possibilities, challenges and use of nanoparticles as a combinatorial approach for enhanced vaccine immunogenicity.

INTRODUCTION: Vaccination is one of the greatest breakthroughs of modern preventative medicine. Despite this, there remain problems surrounding delivery, efficacy and compliance. Thus, there is a pressing need to develop cost-effective vaccine delivery systems that could expand the use of vaccines, particularly within developing countries. Microneedle (MN) arrays, given their ease of use, painlessness and ability to target skin antigen presenting cells, provide an attractive platform for improved vaccine delivery and efficacy. Studies have demonstrated enhanced immunogenicity with the use of MN in comparison to conventional needle. More recently, dissolving MN have been used for efficient delivery of nanoparticles (NP), as a means to enhance antigen immunogenicity. AREAS COVERED: This review introduces the fields of MN technology and nanotechnology, highlighting the recent advances which have been made with these two technologies combined for enhanced vaccine delivery and efficacy. Some key questions that remain to be addressed for adoption of MN in a clinical setting are also evaluated. EXPERT OPINION: MN-mediated vaccine delivery holds potential for expanding access to vaccines, with individuals in developing countries likely to be the principal beneficiaries. The combinatorial approach of utilizing MN coupled with NP, provides opportunities to enhance the immunogenicity of vaccine antigens.

Design and characterisation of a dissolving microneedle patch for intradermal vaccination with heat-inactivated bacteria: A proof of concept study.

This work describes the formulation and evaluation of dissolving microneedle patches (MNs) for intradermal delivery of heat-inactivated bacteria. Pseudomonas aeruginosa, strain PA01, was used as a model bacterium. Utilising a simple, cost effective fabrication process, P. aeruginosa was heat-inactivated and formulated into dissolving MNs, fabricated from aqueous blends of 20% w/w poly(methylvinylether/maleic acid). The resultant MNs were of sufficient mechanical strength to consistently penetrate a validated skin model Parafilm M®, inserting to a depth of between 254 and 381 µm. MNs were successfully inserted into murine skin and partially dissolved. Analysis of MN dissolution kinetics in murine ears via optical coherence tomography showed almost complete MN dissolution 5 min post-insertion. Mice were vaccinated using these optimised MNs by application of one MN to the dorsal surface of each ear (5 min). Mice were subsequently challenged intranasally (24 h) with a live culture of P. aeruginosa (2 × 106 colony forming units). Bacterial load in the lungs of mice vaccinated with P. aeruginosa MNs was significantly (p = 0.0059) lower than those of their unvaccinated counterparts. This proof of concept work demonstrates the potential of dissolving MNs for intradermal vaccination with heat-inactivated bacteria. MNs may be a cost effective, potentially viable delivery system, which could easily be implemented in developing countries, allowing a rapid and simplified approach to vaccinating against a specific pathogen.

Cytokine Signaling Protein 3 Deficiency in Myeloid Cells Promotes Retinal Degeneration and Angiogenesis through Arginase-1 Up-Regulation in Experimental Autoimmune Uveoretinitis.

The suppressor of cytokine signaling protein 3 (SOCS3) critically controls immune cell activation, although its role in macrophage polarization and function remains controversial. Using experimental autoimmune uveoretinitis (EAU) as a model, we show that inflammation-mediated retinal degeneration is exaggerated and retinal angiogenesis is accelerated in mice with SOCS3 deficiency in myeloid cells (LysMCre/+SOCS3fl/fl). At the acute stage of EAU, the population of infiltrating neutrophils was increased and the population of macrophages decreased in LysMCre/+SOCS3fl/fl mice compared with that in wild-type (WT) mice. Real-time RT-PCR showed that the expression of tumor necrosis factor-α, IL-1ß, interferon-γ, granulocyte-macrophage colony-stimulating factor, and arginase-1 was significantly higher in the LysMCre/+SOCS3fl/fl EAU retina in contrast to the WT EAU retina. The percentage of arginase-1+ infiltrating cells was significantly higher in the LysMCre/+SOCS3fl/fl EAU retina than that in the WT EAU retina. In addition, bone marrow-derived macrophages and neutrophils from the LysMCre/+SOCS3fl/fl mice express significantly higher levels of chemokine (C-C motif) ligand 2 and arginase-1 compared with those from WT mice. Inhibition of arginase using an l-arginine analog amino-2-borono-6-hexanoic suppressed inflammation-induced retinal angiogenesis without affecting the severity of inflammation. Our results suggest that SOCS3 critically controls the phenotype and function of macrophages and neutrophils under inflammatory conditions and loss of SOCS3 promotes the angiogenic phenotype of the cells through up-regulation of arginase-1.

Mesenchymal Stromal Cells Modulate Macrophages in Clinically Relevant Lung Injury Models by Extracellular Vesicle Mitochondrial Transfer.

RATIONALE: Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer. OBJECTIVES: To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects. METHODS: Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice. MEASUREMENTS AND MAIN RESULTS: MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSC-derived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects. CONCLUSIONS: In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSC-derived EVs ameliorate lung injury in vivo.

Repeat application of microneedles does not alter skin appearance or barrier function and causes no measurable disturbance of serum biomarkers of infection, inflammation or immunity in mice in vivo.

We address, for the first time, the impact of skin insertion on multiple occasions of polymeric microneedle arrays in an animal model in vivo. Dissolving microneedle arrays prepared from aqueous blends of 20% w/w Gantrez® S-97 BF and 40% w/w poly(vinyl pyrrolidone) 58kDa and hydrogel-forming microneedle arrays prepared from aqueous blends of and poly(ethyleneglycol) 10kDa were repeatedly applied to the skin of hairless mice in vivo. Skin appearance and skin barrier function, as illustrated by measurement of transepidermal water loss, were not measurably altered during the entire study period. Biomarkers of infection, immunity and inflammation/irritation were also statistically unchanged, regardless of the microneedle formulation, needle density or number of applications. Mice remained healthy throughout and continued to gain weight during the study. For example, transepidermal water loss values were typically in the range 10-15gm-2h-1 immediately prior to microneedle insertion and 15-25gm-2h-1 immediately following microneedle removal, regardless of when they were measured during the study periods. Serum biomarker levels, measured immediately post-mortem were always in the range 10-20µgml-1 for C-reactive protein, 0.5-1.5mgml-1 for Immunoglobulin G and 1000-2500pgml-1 for interleukin 1-ß and were never statistically different from untreated controls. No measurable levels of tumour necrosis factor-α were found in any animals. These findings are encouraging for the formulations investigated, suggesting that their repeated use by patients will not cause undesirable side-effects. By beginning to address potential regulatory questions at an early stage, the microneedles field will be ideally-placed to take advantage of the potential market. This work illustrates a potential pre-clinical strategy for development of regulatory dossiers on microneedle technologies.

In vivo studies investigating biodistribution of nanoparticle-encapsulated rhodamine B delivered via dissolving microneedles.

Nanoparticles (NPs) have undergone extensive investigation as drug delivery and targeting vehicles. NP delivery is often via the parenteral route, reliant on administration using hypodermic needles, which can be associated with patient compliance issues and safety concerns. In the recent past, the intradermal delivery of NPs, via novel dissolving microneedle (MN) arrays has garnered interest in the pharmaceutical community. However, published studies using this combinatorial approach have been limited, in that they have focussed on the use of in vitro and ex vivo models only. The current study was designed to answer the fundamental question of how such NPs are distributed in an in vivo murine model, following MN-mediated delivery. Rhodamine B (RhB) was employed as a model tracer dye to facilitate study of biodistribution. Following MN application, RhB was detected in the livers, kidneys, spleens and superficial parotid lymph nodes of the mice. Uptake into the lymphatics was of particular note, as it points towards the potential for utilisation of a minimally-invasive MN delivery strategy in controlled targeting of active drug substances and vaccines to the lymphatics. The use of such a delivery system could, following further development, have far-reaching benefits in enhancement of immunomodulatory and anti-cancer therapies. As a consequence, further investigation of MN/NP combinatorial delivery strategies is warranted.

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.

DNA vaccination for cervical cancer; a novel technology platform of RALA mediated gene delivery via polymeric microneedles.

HPV subtypes (16, 18) are associated with the development of cervical cancer, with oncoproteins E6 and E7 responsible for pathogenesis. The goal of this study was to evaluate our 'smart system' technology platform for DNA vaccination against cervical cancer. The vaccination platform brings together two main components; a peptide RALA which condenses DNA into cationic nanoparticles (NPs), and a polymeric polyvinylpyrrolidone (PVP) microneedle (MN) patch for cutaneous delivery of the loaded NPs. RALA condensed E6/E7 DNA into NPs not exceeding 100nm in diameter, and afforded the DNA protection from degradation in PVP. Sera from mice vaccinated with MN/RALA-E6/E7 were richer in E6/E7-specific IgGs, displayed a greater T-cell-mediated TC-1 cytotoxicity and contained more IFN-γ than sera from mice that received NPs intramuscularly. More importantly, MN/RALA-E6/E7 delayed TC-1 tumor initiation in a prophylactic model, and slowed tumor growth in a therapeutic model of vaccination, and was more potent than intramuscular vaccination.

Reduced epithelial suppressor of cytokine signalling 1 in severe eosinophilic asthma.

Severe asthma represents a major unmet clinical need. Eosinophilic inflammation persists in the airways of many patients with uncontrolled asthma, despite high-dose inhaled corticosteroid therapy. Suppressors of cytokine signalling (SOCS) are a family of molecules involved in the regulation of cytokine signalling via inhibition of the Janus kinase-signal transducers and activators of transcription pathway. We examined SOCS expression in the airways of asthma patients and investigated whether this is associated with persistent eosinophilia.Healthy controls, mild/moderate asthmatics and severe asthmatics were studied. Whole genome expression profiling, quantitative PCR and immunohistochemical analysis were used to examine expression of SOCS1, SOCS2 and SOCS3 in bronchial biopsies. Bronchial epithelial cells were utilised to examine the role of SOCS1 in regulating interleukin (IL)-13 signalling in vitroSOCS1 gene expression was significantly lower in the airways of severe asthmatics compared with mild/moderate asthmatics, and was inversely associated with airway eosinophilia and other measures of T-helper type 2 (Th2) inflammation. Immunohistochemistry demonstrated SOCS1 was predominantly localised to the bronchial epithelium. SOCS1 overexpression inhibited IL-13-mediated chemokine ligand (CCL) 26 (eotaxin-3) mRNA expression in bronchial epithelial cells.Severe asthma patients with persistent airway eosinophilia and Th2 inflammation have reduced airway epithelial SOCS1 expression. SOCS1 inhibits epithelial IL-13 signalling, supporting its key role in regulating Th2-driven eosinophilia in severe asthma.

Correction: Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation.

[This corrects the article DOI: 10.1371/journal.ppat.1004627.].

Mitochondrial Transfer via Tunneling Nanotubes is an Important Mechanism by Which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the In Vitro and In Vivo Models of ARDS.

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte-derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210-2223.

Innate Lymphoid Cells Are the Predominant Source of IL-17A during the Early Pathogenesis of Acute Respiratory Distress Syndrome.

RATIONALE: IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Although IL-17A is the archetypal cytokine of T-helper 17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown. OBJECTIVES: To identify the cellular source and the role of IL-17A in the early phase of lung injury. METHODS: Lung injury was induced in wild-type (C57BL/6) and IL-17 knockout (KO) mice with aerosolized LPS (100 µg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was performed by flow cytometry. MEASUREMENTS AND MAIN RESULTS: A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared with wild-type mice. The majority of RORγt(+) cells in the mouse lung were the recently identified group 3 innate lymphoid cells (ILC3s). Detailed characterization revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in recombinase-activating gene 2 KO mice, which lack T cells but retain innate lymphoid cells. No amelioration of pathology was observed in the recombinase-activating gene 2 KO mice. CONCLUSIONS: IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3s. Modulation of the activity of pILC3s may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.

Targeting Siglecs with a sialic acid-decorated nanoparticle abrogates inflammation.

Sepsis is the most frequent cause of death in hospitalized patients, and severe sepsis is a leading contributory factor to acute respiratory distress syndrome (ARDS). At present, there is no effective treatment for these conditions, and care is primarily supportive. Murine sialic acid-binding immunoglobulin-like lectin-E (Siglec-E) and its human orthologs Siglec-7 and Siglec-9 are immunomodulatory receptors found predominantly on hematopoietic cells. These receptors are important negative regulators of acute inflammatory responses and are potential targets for the treatment of sepsis and ARDS. We describe a Siglec-targeting platform consisting of poly(lactic-co-glycolic acid) nanoparticles decorated with a natural Siglec ligand, di(α2→8) N-acetylneuraminic acid (α2,8 NANA-NP). This nanoparticle induced enhanced oligomerization of the murine Siglec-E receptor on the surface of macrophages, unlike the free α2,8 NANA ligand. Furthermore, treatment of murine macrophages with these nanoparticles blocked the production of lipopolysaccharide-induced inflammatory cytokines in a Siglec-E-dependent manner. The nanoparticles were also therapeutically beneficial in vivo in both systemic and pulmonary murine models replicating inflammatory features of sepsis and ARDS. Moreover, we confirmed the anti-inflammatory effect of these nanoparticles on human monocytes and macrophages in vitro and in a human ex vivo lung perfusion (EVLP) model of lung injury. We also established that interleukin-10 (IL-10) induced Siglec-E expression and α2,8 NANA-NP further augmented the expression of IL-10. Indeed, the effectiveness of the nanoparticle depended on IL-10. Collectively, these results demonstrated a therapeutic effect of targeting Siglec receptors with a nanoparticle-based platform under inflammatory conditions.