Treatment with inhaled aerosolised ethanol reduces viral load and potentiates macrophage responses in an established influenza mouse model.

AIM: Treatment options for viral lung infections are currently limited. We aimed to explore the safety and efficacy of inhaled ethanol in an influenza-infection mouse model. MATERIALS AND METHODS: In a safety and tolerability experiment, 80 healthy female BALB/c mice (20 per group) were exposed to nebulized saline (control) or three concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods, with a two-hour break between exposures. In a separate subsequent experiment, 40 Female BALB/c mice were nasally inoculated with 104.5 plaque-forming units of immediate virulence "Mem71" influenza. Infection was established for 48-h before commencing treatment in 4 groups of 10 mice with either nebulized saline (control) or one of 3 different concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods daily over three consecutive days. In both experiments, mouse behavior, clinical scores, weight change, bronchoalveolar lavage cell viability, cellular composition, and cytokine levels, were assessed 24-h following the final exposure, with viral load also assessed after the second experiment. RESULTS: In uninfected BALB/c mice, 3x30-minute exposures to nebulized 40%, 60%, and 80% ethanol resulted in no significant differences in mouse weights, cell counts/viability, cytokines, or morphometry measures. In Mem71-influenza infected mice, we observed a dose-dependent reduction in viral load in the 80%-treated group and potentiation of macrophage numbers in the 60%- and 80%-treated groups, with no safety concerns. CONCLUSIONS: Our data provides support for inhaled ethanol as a candidate treatment for respiratory infections.

An infant mouse model of influenza-driven nontypeable Haemophilus influenzae colonization and acute otitis media suitable for preclinical testing of novel therapies.

Nontypeable Haemophilus influenzae (NTHi) is a major otitis media (OM) pathogen, with colonization a prerequisite for disease development. Most acute OM is in children <5 years old, with recurrent and chronic OM impacting hearing and learning. Therapies to prevent NTHi colonization and/or disease are needed, especially for young children. Respiratory viruses are implicated in driving the development of bacterial OM in children. We have developed an infant mouse model of influenza-driven NTHi OM, as a preclinical tool for the evaluation of safety and efficacy of clinical therapies to prevent NTHi colonization and the development of OM. In this model, 100% of infant BALB/cARC mice were colonized with NTHi, and all developed NTHi OM. Influenza A virus (IAV) facilitated the establishment of dense (1 × 105 CFU/mL) and long-lasting (6 days) NTHi colonization. IAV was essential for the development of NTHi OM, with 100% of mice in the IAV/NTHi group developing NTHi OM compared with 8% of mice in the NTHi only group. Histological analysis and cytokine measurements revealed that the inflammation observed in the middle ear of the infant mice with OM reflected inflammation observed in children with OM. We have developed the first infant mouse model of NTHi colonization and OM. This ascension model uses influenza-driven establishment of OM and reflects the clinical pathology of bacterial OM developing after a respiratory virus infection. This model provides a valuable tool for testing therapies to prevent or treat NTHi colonization and disease in young children.

Nasal Delivery of Haemophilus haemolyticus Is Safe, Reduces Influenza Severity, and Prevents Development of Otitis Media in Mice.

BACKGROUND: Despite vaccination, influenza and otitis media (OM) remain leading causes of illness. We previously found that the human respiratory commensal Haemophilus haemolyticus prevents bacterial infection in vitro and that the related murine commensal Muribacter muris delays OM development in mice. The observation that M muris pretreatment reduced lung influenza titer and inflammation suggests that these bacteria could be exploited for protection against influenza/OM. METHODS: Safety and efficacy of intranasal H haemolyticus at 5 × 107 colony-forming units (CFU) was tested in female BALB/cARC mice using an influenza model and influenza-driven nontypeable Haemophilus influenzae (NTHi) OM model. Weight, symptoms, viral/bacterial levels, and immune responses were measured. RESULTS: Intranasal delivery of H haemolyticus was safe and reduced severity of influenza, with quicker recovery, reduced inflammation, and lower lung influenza virus titers (up to 8-fold decrease vs placebo; P ≤ .01). Haemophilus haemolyticus reduced NTHi colonization density (day 5 median NTHi CFU/mL = 1.79 × 103 in treatment group vs 4.04 × 104 in placebo, P = .041; day 7 median NTHi CFU/mL = 28.18 vs 1.03 × 104; P = .028) and prevented OM (17% OM in treatment group, 83% in placebo group; P = .015). CONCLUSIONS: Haemophilus haemolyticus has potential as a live biotherapeutic for prevention or early treatment of influenza and influenza-driven NTHi OM. Additional studies will deem whether these findings translate to humans and other respiratory infections.

In Vivo Evidence of Respiratory Syncytial Virus Persistence in a Subset of Pulmonary Dendritic Cells Following a Primary Infection.

Respiratory syncytial virus (RSV) causes annual epidemics of infections affecting the whole population. In vitro, it has been shown to infect and persist in human dendritic cells (DCs) for prolonged periods. Initially persistence is associated with low levels of replication before the virus becomes dormant. Reactivation of viral replication can be triggered many months later. Infection of DCs is likely to influence the host's ability to generate effective long-term memory responses. A well-established animal was utilized to confirm that RSV both infects and persists in pulmonary DCs in vivo. Mice were infected with a modified strain of RSV expressing red fluorescent protein (RSV-RFP) when replicating. Clinical symptoms of infection were monitored using weight change and inflammatory cell counts from bronchoalveolar lavage, which correlated with the RSV viral titer (quantitative polymerase chain reaction). Lung tissues were collected at 3, 5, 7, and 21 days postinfection (dpi) to assess leukocyte populations by flow cytometry. Clinical symptoms and RSV viral load peaked at 5 dpi. RSV-RFP was most prevalent in macrophages at 3 dpi and also observed in B cells and DCs. At 21 dpi, RSV-RFP remained evident in a subset of conventional DCs (CD103+CD11b+) even though both clinical symptoms and pulmonary inflammation had resolved. These results confirm that in this well-established mouse model, RSV persists in lung conventional DCs following resolution of the acute infection. Further work is required to explore whether the virus continues with low-level replication before becoming dormant in vivo, as has been described in vitro.

Occurrence of methicillin-resistant Staphylococcus aureus in pets and their owners in rural and urban communities in Trinidad.

INTRODUCTION: The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in pets and their owners has increased due to the misuse and abuse of antibiotics. This study compared the prevalence of MRSA and Staphylococcus aureus strains in pets and their owners in urban and rural communities in Trinidad. METHODOLOGY: Questionnaires were administered to gather demographic and risk factor data for MRSA for human participants, and their pets. Nasal swabs were obtained from 100 pets (dogs and cats) and their human owners. For the isolation of MRSA, nasal swabs obtained were enriched and then plated on selective media. Staphylococcus aureus was identified using standard biochemical procedures. The resistance of S. aureus initially assessed detection of MRSA isolates to cefoxitin and confirmed by the PBP2a latex agglutination test. Antibiotic resistance was determined using the disc diffusion method. RESULTS: The prevalence of MRSA was 6.0% (3/50) and 2.0% (1/50) in household pet animals and their owners, respectively in urban communities, while in rural communities, the prevalence was 6.0% (3/50) and 12.0% (6/50) respectively. The prevalence of S. aureus in pet owners was higher in the rural community (44.0%) compared to urban (30.0%). However, in pet animals, S. aureus was more frequently isolated from urban communities (78.0%) than rural ones (66.0%). Amongst the S. aureus isolates, 81.7% were resistant to one or more antimicrobial agents. CONCLUSIONS: This study has demonstrated that living in a rural community increased the odds of MRSA colonization.

Tocilizumab for the treatment of giant cell arteritis in Scotland: a report on behalf of the Scottish Society for Rheumatology standards subgroup.

Objectives: The aim was to describe a modern National Health Service (NHS) Scotland cohort of patients with GCA over 12 months of care to include clinical presentation, practices relating to assessment and treatment, and specifically, the use of tocilizumab. Methods: A multicentre audit of patients newly diagnosed with GCA between November 2019 and October 2021 was established on behalf of the Scottish Society for Rheumatology. Clinical data were collected retrospectively by rheumatology teams at participating NHS centres using electronic patient records. An extended cohort of patients from NHS Lothian was examined to investigate outcomes of tocilizumab use for >1 year. Results: Sixty-three patients from three NHS Scotland health boards were included, with analysis of data from 216 clinic episodes. Mean follow-up was 371 days. Mean age was 71 years; 62% were female. The most common presenting features were headache (93.6%), scalp tenderness (82.5%) and ocular symptoms (24%). At baseline, 63% of patients had at least one existing risk factor for adverse outcomes from high-dose CS use, namely hypertension (57.1%), diabetes (24%) and osteoporosis (11%). Thirty per cent of all patients (19 of 63) received tocilizumab, with only 11% (7 of 63) receiving tocilizumab owing to glucocorticoid risk factors at baseline. One-quarter of all patients (16 of 63) experienced relapse of GCA during follow-up, of whom six were subsequently treated with tocilizumab. Conclusion: This multicentre audit demonstrates that despite its availability for patients with risk factors for CS adversity and those who suffer relapse of GCA, tocilizumab is used in less than one-quarter of patients who might benefit. The reasons for this require further exploration.

Protection against neonatal respiratory viral infection via maternal treatment during pregnancy with the benign immune training agent OM-85.

OBJECTIVES: Incomplete maturation of immune regulatory functions at birth is antecedent to the heightened risk for severe respiratory infections during infancy. Our forerunner animal model studies demonstrated that maternal treatment with the microbial-derived immune training agent OM-85 during pregnancy promotes accelerated postnatal maturation of mechanisms that regulate inflammatory processes in the offspring airways. Here, we aimed to provide proof of concept for a novel solution to reduce the burden and potential long-term sequelae of severe early-life respiratory viral infection through maternal oral treatment during pregnancy with OM-85, already in widespread human clinical use. METHODS: In this study, we performed flow cytometry and targeted gene expression (RT-qPCR) analysis on lungs from neonatal offspring whose mothers received oral OM-85 treatment during pregnancy. We next determined whether neonatal offspring from OM-85 treated mothers demonstrate enhanced protection against lethal lower respiratory infection with mouse-adapted rhinovirus (vMC0), and associated lung immune changes. RESULTS: Offspring from mothers treated with OM-85 during pregnancy display accelerated postnatal seeding of lung myeloid populations demonstrating upregulation of function-associated markers. Offspring from OM-85 mothers additionally exhibit enhanced expression of TLR4/7 and the IL-1ß/NLRP3 inflammasome complex within the lung. These treatment effects were associated with enhanced capacity to clear an otherwise lethal respiratory viral infection during the neonatal period, with concomitant regulation of viral-induced IFN response intensity. CONCLUSION: These results demonstrate that maternal OM-85 treatment protects offspring against lethal neonatal respiratory viral infection by accelerating development of innate immune mechanisms crucial for maintenance of local immune homeostasis in the face of pathogen challenge.

Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice.

Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus, a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 105 CFU/ml to 9 × 103 CFU/ml (P = 0.0004). Only 1/12 M. muris-pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris-pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.

Transplacental Innate Immune Training via Maternal Microbial Exposure: Role of XBP1-ERN1 Axis in Dendritic Cell Precursor Programming.

We recently reported that offspring of mice treated during pregnancy with the microbial-derived immunomodulator OM-85 manifest striking resistance to allergic airways inflammation, and localized the potential treatment target to fetal conventional dendritic cell (cDC) progenitors. Here, we profile maternal OM-85 treatment-associated transcriptomic signatures in fetal bone marrow, and identify a series of immunometabolic pathways which provide essential metabolites for accelerated myelopoiesis. Additionally, the cDC progenitor compartment displayed treatment-associated activation of the XBP1-ERN1 signalling axis which has been shown to be crucial for tissue survival of cDC, particularly within the lungs. Our forerunner studies indicate uniquely rapid turnover of airway mucosal cDCs at baseline, with further large-scale upregulation of population dynamics during aeroallergen and/or pathogen challenge. We suggest that enhanced capacity for XBP1-ERN1-dependent cDC survival within the airway mucosal tissue microenvironment may be a crucial element of OM-85-mediated transplacental innate immune training which results in postnatal resistance to airway inflammatory disease.

Early life ovalbumin sensitization and aerosol challenge for the induction ofallergic airway inflammation in a BALB/c murine model.

The early life period represents a time of immunological plasticity whereby the functionally immature immune system is highly susceptible to environmental stimulation. Perennial aeroallergen and respiratory viral infection induced sporadic episodes of lung inflammation during this temporal window represent major risk factors for initiation of allergic asthmatic disease. Murine models are widely used as an investigative tool to examine the pathophysiology of allergic asthma; however, models in current usage typically do not encapsulate the early life period which represents the time of maximal risk for disease inception in humans. To address this issue, this protocol adapted an experimental animal model of disease for sensitization to ovalbumin during the immediate post-weaning period beginning at 21 days of age. By initially sensitizing mice during this early life post-weaning period, researchers can more closely align experimental allergic airway disease models with the human age group most at risk for asthma development.

Quantification of serum ovalbumin-specific immunoglobulin E titrevia in vivo passive cutaneous anaphylaxis assay.

Murine models of allergic airway disease are frequently used as a tool to elucidate the cellular and molecular mechanisms of tissue-specific asthmatic disease pathogenesis. Paramount to the success of these models is the induction of experimental antigen sensitization, as indicated by the presence of antigen-specific serum immunoglobulin E. The quantification of antigen-specific serum IgE is routinely performed via enzyme-linked immunosorbent assay. However, the reproducibility of these in vitro assays can vary dramatically in our experience. Furthermore, quantifying IgE via in vitro methodologies does not enable the functional relevance of circulating IgE levels to be considered. As a biologically appropriate alternative method, we describe herein a highly reproducible in vivo passive cutaneous anaphylaxis assay using Sprague Dawley rats for the quantification of ovalbumin-specific IgE in serum samples from ovalbumin-sensitized murine models. Briefly, this in vivo assay involves subcutaneous injections of serum samples on the back of a Sprague Dawley rat, followed 24 h later by intravenous injection of ovalbumin and a blue detection dye. The subsequent result of antigen-IgE mediated inflammation and leakage of blue dye into the initial injection site indicates the presence of ovalbumin-specific IgE within the corresponding serum sample.

Pregnancy Induces a Steady-State Shift in Alveolar Macrophage M1/M2 Phenotype That Is Associated With a Heightened Severity of Influenza Virus Infection: Mechanistic Insight Using Mouse Models.

BACKGROUND: Influenza virus infection during pregnancy is associated with enhanced disease severity. However, the underlying mechanisms are still not fully understood. We hypothesized that normal alveolar macrophage (AM) functions, which are central to maintaining lung immune homeostasis, are altered during pregnancy and that this dysregulation contributes to the increased inflammatory response to influenza virus infection. METHODS: Time-mated BALB/c mice were infected with a low dose of H1N1 influenza A virus at gestation day 9.5. Inflammatory cells in bronchoalveolar lavage (BAL) fluid were assessed by flow cytometry. RESULTS: Our findings confirm previous reports of increased severity of influenza virus infection in pregnant mice. The heightened inflammatory response detected in BAL fluid from infected pregnant mice was characterized by neutrophil-rich inflammation with concomitantly reduced numbers of AM, which were slower to return to baseline counts, compared with nonpregnant infected mice. The increased infection severity and inflammatory responses to influenza during pregnancy were associated with a pregnancy-induced shift in AM phenotype at homeostatic baseline, from the M1 (ie, classical activation) state toward the M2 (ie, alternative activation) state, as evidence by increased expression of CD301 and reduced levels of CCR7. CONCLUSION: These results show that pregnancy is associated with an alternatively activated phenotype of AM before infection, which may contribute to heightened disease severity.

Transplacental immune modulation with a bacterial-derived agent protects against allergic airway inflammation.

Chronic allergic inflammatory diseases are a major cause of morbidity, with allergic asthma alone affecting over 300 million people worldwide. Epidemiological studies demonstrate that environmental stimuli are associated with either the promotion or prevention of disease. Major reductions in asthma prevalence are documented in European and US farming communities. Protection is associated with exposure of mothers during pregnancy to microbial breakdown products present in farm dusts and unprocessed foods and enhancement of innate immune competence in the children. We sought to develop a scientific rationale for progressing these findings toward clinical application for primary disease prevention. Treatment of pregnant mice with a defined, clinically approved immune modulator was shown to markedly reduce susceptibility of their offspring to development of the hallmark clinical features of allergic airway inflammatory disease. Mechanistically, offspring displayed enhanced dendritic cell-dependent airway mucosal immune surveillance function, which resulted in more efficient generation of mucosal-homing regulatory T cells in response to local inflammatory challenge. We provide evidence that the principal target for maternal treatment effects was the fetal dendritic cell progenitor compartment, equipping the offspring for accelerated functional maturation of the airway mucosal dendritic cell network following birth. These data provide proof of concept supporting the rationale for developing transplacental immune reprogramming approaches for primary disease prevention.

Patient Values and Preferences Regarding Opioids for Chronic Noncancer Pain: A Systematic Review.

Objective: Shared-care decision-making between patients and clinicians involves making trade-offs between desirable and undesirable consequences of management strategies. Although patient values and preferences should provide the basis for these trade-offs, few guidelines consider the relevant evidence when formulating recommendations. To inform a guideline for use of opioids in patients with chronic noncancer pain, we conducted a systematic review of studies exploring values and preferences of affected patients toward opioid therapy. Methods: We searched MEDLINE, CINAHL, EMBASE, and PsycINFO from the inception of each database through October 2016. We included studies examining patient preferences for alternative approaches to managing chronic noncancer pain and studies that assessed how opioid-using chronic noncancer pain patients value alternative health states and their experiences with treatment. We compiled structured summaries of the results. Results: Pain relief and nausea and vomiting were ranked as highly significant outcomes across studies. When considered, the adverse effect of personality changes was rated as equally important. Constipation was assessed in most studies and was an important outcome, secondary to pain relief and nausea and vomiting. Of only two studies that evaluated addiction, both found it less important to patients than pain relief. No studies examined opioid overdose, death, or diversion. Conclusion: Our findings suggest that the adverse effects of opioids, especially nausea and vomiting, may reduce or eliminate any net benefit of opioid therapy unless pain relief is significant (>2 points on a 10-point scale). Further research should investigate patient values and preferences regarding opioid overdose, diversion, and death.

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and Ultraviolet-Chimeric Mice.

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.

PGE2 pulsing of murine bone marrow cells reduces migration of daughter monocytes/macrophages in vitro and in vivo.

Monocytes/macrophages differentiating from bone marrow (BM) cells pulsed for 2 hours at 37°C with a stabilized derivative of prostaglandin E2, 16,16-dimethyl PGE2 (dmPGE2), migrated less efficiently toward a chemoattractant than monocytes/macrophages differentiated from BM cells pulsed with vehicle. To confirm that the effect on BM cells was long lasting and to replicate human BM transplantation, chimeric mice were established with donor BM cells pulsed for 2 hours with dmPGE2 before injection into marrow-ablated congenic recipient mice. After 12 weeks, when high levels (90%) of engraftment were obtained, regenerated BM-derived monocytes/macrophages differentiating in vitro or in vivo migrated inefficiently toward the chemokines colony-stimulating factor-1 (CSF-1) and chemokine (C-C motif) ligand 2 (CCL2) or thioglycollate, respectively. Our results reveal long-lasting changes to progenitor cells of monocytes/macrophages by a 2-hour dmPGE2 pulse that, in turn, limits the migration of their daughter cells to chemoattractants and inflammatory mediators.

UV Irradiation of Skin Enhances Glycolytic Flux and Reduces Migration Capabilities in Bone Marrow-Differentiated Dendritic Cells.

A systemic immunosuppression follows UV irradiation of the skin of humans and mice. In this study, dendritic cells (DCs) differentiating from the bone marrow (BM) of UV-irradiated mice had a reduced ability to migrate toward the chemokine (C-C motif) ligand 21. Fewer DCs also accumulated in the peritoneal cavity of UV-chimeric mice (ie, mice transplanted with BM from UV-irradiated mice) after injection of an inflammatory stimulus into that site. We hypothesized that different metabolic states underpin altered DC motility. Compared with DCs from the BM of nonirradiated mice, those from UV-irradiated mice produced more lactate, consumed more glucose, and had greater glycolytic flux in a bioenergetics stress test. Greater expression of 3-hydroxyanthranilate 3,4-dioxygenase was identified as a potential contributor to increased glycolysis. Inhibition of 3-hydroxyanthranilate 3,4-dioxygenase by 6-chloro-dl-tryptophan prevented both increased lactate production and reduced migration toward chemokine (C-C motif) ligand 21 by DCs differentiated from BM of UV-irradiated mice. UV-induced prostaglandin E2 has been implicated as an intermediary in the effects of UV radiation on BM cells. DCs differentiating from BM cells pulsed in vitro for 2 hours with dimethyl prostaglandin E2 were functionally similar to those from the BM of UV-irradiated mice. Reduced migration of DCs to lymph nodes associated with increased glycolytic flux may contribute to their reduced ability to initiate new immune responses in UV-irradiated mice.

Reduced immune responses in chimeric mice engrafted with bone marrow cells from mice with airways inflammation.

OBJECTIVE: During respiratory inflammation, it is generally assumed that dendritic cells differentiating from the bone marrow are immunogenic rather than immunoregulatory. Using chimeric mice, the outcomes of airways inflammation on bone marrow progenitor cells were studied. METHODS: Immune responses were analyzed in chimeric mice engrafted for >16 weeks with bone marrow cells from mice with experimental allergic airways disease (EAAD). RESULTS: Responses to sensitization and challenge with the allergen causing inflammation in the bone marrow-donor mice were significantly reduced in the chimeric mice engrafted with bone marrow cells from mice with EAAD (EAAD-chimeric). Responses to intranasal LPS and topical fluorescein isothiocyanate (non-specific challenges) were significantly attenuated. Fewer activated dendritic cells from the airways and skin of the EAAD-chimeric mice could be tracked to the draining lymph nodes, and may contribute to the significantly reduced antigen/chemical-induced hypertrophy in the draining nodes, and the reduced immune responses to sensitizing allergens. Dendritic cells differentiating in vitro from the bone marrow of >16 weeks reconstituted EAAD-chimeric mice retained an ability to poorly prime immune responses when transferred into naïve mice. CONCLUSIONS: Dendritic cells developing from bone marrow progenitors during airways inflammation are altered such that daughter cells have reduced antigen priming capabilities.

Prostaglandin E2 imprints a long-lasting effect on dendritic cell progenitors in the bone marrow.

Dendritic cells (DCs) that differentiate in vitro from the bone marrow (BM) of mice with prostaglandin E2 (PGE2)-associated inflammation of the skin, airways, or peritoneal cavity poorly initiate immune responses. To remove in vitro differentiation and allow BM-derived DCs to seed the periphery under steady-state conditions, as well as study the molecule proposed responsible, chimeric mice were engrafted for >16 wk with BM cells from mice exposed to PGE2. Serial PGE2-chimeric mice were established with BM cells from the primary chimeric mice. Immune responses in the airways and skin of the PGE2-chimeric mice and serial PGE2-chimeric mice were significantly attenuated. After inflammatory challenges by intranasal LPS, topical fluorescein isothiocyanate, and intraperitoneal alum, DCs, macrophages, and neutrophils trafficked poorly in PGE2-chimeric mice and serial PGE2-chimeric mice. Injection of BM-differentiated DCs from nonchimeric mice restored the reduced immune responses of PGE2-chimeric mice. DCs from BM of 16-wk-engrafted PGE2-chimeric and serial PGE2-chimeric mice resembled cells differentiated from BM exposed to PGE2 for only 3 d, demonstrating the long-lasting effect of PGE2 on DC progenitors. PGE2 attenuates systemic immune responses by modulating myeloid cell progenitors in the BM such that BM-derived, terminally differentiated myeloid cells have poor trafficking ability to sites of need.

Characterization of regulatory dendritic cells differentiated from the bone marrow of UV-irradiated mice.

When antigen-loaded dendritic cells (DCs) differentiated from the bone marrow (BM) of UV-irradiated mice (UV-BMDCs) were adoptively transferred into naive mice or mice pre-sensitized with that antigen, the recipients exhibited a reduced immune response following antigen challenge. Hence, UV-BMDCs are poorly immunogenic and can suppress pre-existing immunity. The UV-induced effect on BM-derived DCs was rapid (observed 1 day after UV radiation), long-lasting (observed 10 days after UV radiation) and UV dose-dependent. The mechanism by which UV-BMDCs could regulate immunity was investigated. The CD11c(+) cells, differentiated using granulocyte-macrophage colony-stimulating factor + interleukin-4, were confirmed to be DCs because they did not express the myeloid-derived suppressor cell marker, Gr1. UV-BMDCs did not display altered antigen uptake, processing or ability to activate T cells in vitro. When gene expression in UV-BMDCs and DCs differentiated from the BM of non-irradiated mice (control-BMDCs) was examined, Ccl7, Ccl8 and CSF1R (CD115) mRNA transcripts were up-regulated in UV-BMDCs compared with control-BMDCs. However, neutralizing antibodies for Ccl7 and Ccl8 did not abrogate the reduced immunogenicity of UV-BMDCs in vivo. Moreover, the up-regulation of CSF1R transcript did not correspond with increased receptor expression on UV-BMDCs. The phenotypes of UV-BMDCs and control-BMDCs were similar, with no difference in the expression of CD4, CD8α, CD103, B220 or F4/80, or the regulatory molecules CCR7 (CD197), FasL (CD95L), B7H3 (CD276) and B7H4. However, PDL1 (CD274) expression was reduced in UV-BMDCs compared with control-BMDCs following lipopolysaccharide stimulation. In summary, UV-BMDCs do not express the classical phenotypic or gene expression properties of DCs reported by others as 'regulatory' or 'tolerogenic'.