Mouse Model of Sendai Virus-Induced Lung Disease.

Sendai virus (SeV), also known as the murine parainfluenza virus 1, is an enveloped negative-sense RNA paramyxovirus from the family Paramyxoviridae and genus Respirovirus. The virus was named after Sendai, city in Japan, where it was first isolated (Kuroya, Ishida, Yokohama Med Bull 4:217-233, 1953). Antigenically, SeV is closely related to human parainfluenza viruses 1 and 3. SeV is pneumotropic and naturally infects the respiratory tract of rodents. At the proper inoculum (2 × 105 pfu), SeV causes infection that is limited to the airway mucosa and inflammation mainly restricted to bronchiolar tissues as seen in asthma pathogenesis models using C57BL/6 wild-type mice (Walter et al, J Clin Invest 110:165-175, 2002). We utilize SeV to explore the mechanism(s) by which a respiratory viral infection translates into postviral airway disease in mice. This chapter primarily describes the protocols we use to infect mice in vivo, assay viral replication, and assess outcomes in the lungs of the host.

Type II alveolar cell MHCII improves respiratory viral disease outcomes while exhibiting limited antigen presentation.

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

Neutrophils in respiratory viral infections.

Viral respiratory infections are a common cause of severe disease, especially in infants, people who are immunocompromised, and in the elderly. Neutrophils, an important innate immune cell, infiltrate the lungs rapidly after an inflammatory insult. The most well-characterized effector mechanisms by which neutrophils contribute to host defense are largely extracellular and the involvement of neutrophils in protection from numerous bacterial and fungal infections is well established. However, the role of neutrophils in responses to viruses, which replicate intracellularly, has been less studied. It remains unclear whether and, by which underlying immunological mechanisms, neutrophils contribute to viral control or confer protection against an intracellular pathogen. Furthermore, neutrophils need to be tightly regulated to avoid bystander damage to host tissues. This is especially relevant in the lung where damage to delicate alveolar structures can compromise gas exchange with life-threatening consequences. It is inherently less clear how neutrophils can contribute to host immunity to viruses without causing immunopathology and/or exacerbating disease severity. In this review, we summarize and discuss the current understanding of how neutrophils in the lung direct immune responses to viruses, control viral replication and spread, and cause pathology during respiratory viral infections.

Toll-Interacting Protein in Pulmonary Diseases. Abiding by the Goldilocks Principle.

TOLLIP (Toll-interacting protein) is an intracellular adaptor protein with diverse actions throughout the body. In a context- and cell type-specific manner, TOLLIP can function as an inhibitor of inflammation and endoplasmic-reticulum stress, an activator of autophagy, or a critical regulator of intracellular vacuole trafficking. The distinct functions of this protein have been linked to innate immune responses and lung epithelial-cell apoptosis. TOLLIP genetic variants have been associated with a variety of chronic lung diseases, including idiopathic pulmonary fibrosis, asthma, and primary graft dysfunction after lung transplantation, and with infections, such as tuberculosis, Legionella pneumonia, and respiratory viruses. TOLLIP exists in a delicate homeostatic balance, with both positive and negative effects on the trajectory of pulmonary diseases. This translational review summarizes the genetic and molecular associations that link TOLLIP to the development and progression of noninfectious and infectious pulmonary diseases. We highlight current limitations of in vitro and in vivo models in assessing the role of TOLLIP in these conditions, and we describe future approaches that will enable a more nuanced exploration of the role of TOLLIP in pulmonary conditions. There has been a surge in recent research evaluating the role of this protein in human diseases, but critical mechanistic pathways require further exploration. By understanding its biologic functions in disease-specific contexts, we will be able to determine whether TOLLIP can be therapeutically modulated to treat pulmonary diseases.

Leveraging 3D Model Systems to Understand Viral Interactions with the Respiratory Mucosa.

Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus-host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air-liquid interface, organoids, or 'on-chip' technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium-the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.

Infection of calves with in-vivo passaged bovine parainfluenza-3 virus, alone or in combination with bovine respiratory syncytial virus and bovine coronavirus.

Bovine respiratory disease complex is etiologically complex and usually involves co-infection by several agents, including bovine parainfluenza virus-3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine coronavirus (BCoV). Traditionally, vaccines have been tested in seronegative calves infected with a single in vitro-passaged agent, often with little disease, resulting in unvaccinated subjects. To overcome the potential problem of attenuation coincident with in vitro culture of the viruses, cocktails of field isolates of BPIV-3s and BCoVs were passaged in the lungs of neonatal colostrum-deprived calves. Lung lavage fluids were used as inocula, alone and in combination with in-vivo passaged BRSV, and aerosolized into a trailer containing conventionally reared 9-week-old weaned Holstein calves with decayed, but still measurable, maternal antibodies. Calves developed acute respiratory disease of variable severity. Upon necropsy, there were characteristic gross and histologic lesions in the respiratory tract, associated immunohistochemically with BPIV-3, BRSV, and BCoV. In-vivo passage of viruses is an alternative to in vitro culture to produce inocula to better study the pathogenesis of infection and more rigorously and relevantly assess vaccine efficacy.

Le complexe des maladies respiratoires bovines possède une étiologie complexe et implique habituellement une co-infection par plusieurs agents, incluant le virus parainfluenza bovin 3 (BPIV-3), le virus respiratoire syncitial bovin (BRSV) et le coronavirus bovin (BCoV). Traditionnellement, les vaccins ont été testés chez des veaux séronégatifs infectés avec un seul agent cultivé in vitro, présentant souvent peu de maladie, résultant en des sujets non-vaccinés. Afin de contrecarrer le problème potentiel d'atténuation associé à la culture in vitro des virus, des cocktails d'isolats de champs de BPIV-3 et de BCoV furent passés dans des poumons de veaux nouveau-nés privés de colostrum. Les liquides de lavage pulmonaire furent utilisés comme inoculum, seul et en combinaison avec des BRSV passés in vivo, et aérosolisés dans une remorque contenant des veaux Holstein sevrés élevés de manière conventionnelle âgés de 9 semaines ayant des anticorps maternels en déclin mais toujours mesurables. Les veaux ont développé une maladie respiratoire aiguë de sévérité variable. Lors de la nécropsie, il y avait des lésions macroscopiques et histologiques caractéristiques dans le tractus respiratoire, associées immuno-histochimiquement avec BPIV-3, BRSV et BCoV. Le passage in vivo de virus est une alternative à la culture in vitro afin de produire un inoculum permettant de mieux étudier la pathogénie de l'infection et d'évaluer plus rigoureusement et plus pertinemment l'efficacité de vaccins.(Traduit par Docteur Serge Messier).

Quantitative single-cell interactomes in normal and virus-infected mouse lungs.

Mammalian organs consist of diverse, intermixed cell types that signal to each other via ligand-receptor interactions - an interactome - to ensure development, homeostasis and injury-repair. Dissecting such intercellular interactions is facilitated by rapidly growing single-cell RNA sequencing (scRNA-seq) data; however, existing computational methods are often not readily adaptable by bench scientists without advanced programming skills. Here, we describe a quantitative intuitive algorithm, coupled with an optimized experimental protocol, to construct and compare interactomes in control and Sendai virus-infected mouse lungs. A minimum of 90 cells per cell type compensates for the known gene dropout issue in scRNA-seq and achieves comparable sensitivity to bulk RNA sequencing. Cell lineage normalization after cell sorting allows cost-efficient representation of cell types of interest. A numeric representation of ligand-receptor interactions identifies, as outliers, known and potentially new interactions as well as changes upon viral infection. Our experimental and computational approaches can be generalized to other organs and human samples.

Two Cases of Primary Human Parainfluenza Virus 1 Pneumonia in Which Bronchoalveolar Lavage Fluid Yielded Human Parainfluenza Virus 1.

Two patients, a 76-year-old woman and 66-year-old woman, presented to our hospital with symptoms of lower respiratory tract infection. Both patients showed chest imaging findings of bilateral ground-glass opacities and consolidations. We initially suspected these patients of having influenza-associated pneumonia and cryptogenic organizing pneumonia, respectively, and performed bronchoalveolar lavage, but only human parainfluenza virus-1 infection was detected by multiplex polymerase chain reaction testing. These findings suggest that pneumonia due to human parainfluenza virus-1 should be included in the differential diagnosis of such cases.

Authentic Modeling of Human Respiratory Virus Infection in Human Pluripotent Stem Cell-Derived Lung Organoids.

Infectious viruses so precisely fit their hosts that the study of natural viral infection depends on host-specific mechanisms that affect viral infection. For human parainfluenza virus 3, a prevalent cause of lower respiratory tract disease in infants, circulating human viruses are genetically different from viruses grown in standard laboratory conditions; the surface glycoproteins that mediate host cell entry on circulating viruses are suited to the environment of the human lung and differ from those of viruses grown in cultured cells. Polarized human airway epithelium cultures have been used to represent the large, proximal airways of mature adult airways. Here we modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids derived from human pluripotent stem cells contain mesoderm and pulmonary endoderm and develop into branching airway and alveolar structures. Whole-genome sequencing analysis of parainfluenza viruses replicating in the organoids showed maintenance of nucleotide identity, suggesting that no selective pressure is exerted on the virus in this tissue. Infection with parainfluenza virus led to viral shedding without morphological changes, while respiratory syncytial virus infection induced detachment and shedding of infected cells into the lung organoid lumens, reminiscent of parainfluenza and respiratory syncytial virus in human infant lungs. Measles virus infection, in contrast, induced syncytium formation. These human stem cell-derived lung organoids may serve as an authentic model for respiratory viral pathogenesis in the developing or infant lung, recapitulating respiratory viral infection in the host.IMPORTANCE Respiratory viruses are among the first pathogens encountered by young children, and the significant impact of these viral infections on the developing lung is poorly understood. Circulating viruses are suited to the environment of the human lung and are different from those of viruses grown in cultured cells. We modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids, derived from human pluripotent stem cells, develop into branching airway and alveolar structures and provide a tissue environment that maintains the authentic viral genome. The lung organoids can be genetically engineered prior to differentiation, thereby generating tissues bearing or lacking specific features that may be relevant to viral infection, a feature that may have utility for the study of host-pathogen interaction for a range of lung pathogens.

Fetal Pathology in an Aborted Holstein Fetus Infected With Bovine Parainfluenza Virus-3 Genotype A.

Bovine parainfluenza virus-3 (BPIV-3) is a recognized respiratory pathogen of cattle, and it has also been identified in aborted fetuses. However, little is known of this agent as a reproductive pathogen and detailed descriptions of fetal pathology on natural cases are lacking in the scientific literature. This article describes and illustrates lesions in a fetus spontaneously aborted by a first-calving Holstein heifer, naturally infected with BPIV-3 genotype A, broadening the current knowledge on fetal pathology by this virus. Fetal autopsy revealed diffusely reddened, rubbery and unexpanded lungs. Histologically, there was necrotizing bronchiolitis/alveolitis with intraluminal fibrin exudate and syncytial cells in the bronchiolar/alveolar spaces, and non-suppurative peribronchiolitis and perivascular interstitial pneumonia. In the small intestine there was multifocal necrotizing cryptitis and occasional necrotic syncytial enterocytes. Intralesional and extralesional BPIV-3 antigen was detected by immunohistochemistry in the lung and small intestine, and BPIV-3a was identified in fetal tissues by RT-PCR and sequencing.

Sendai Virus V Protein Inhibits the Secretion of Interleukin-1ß by Preventing NLRP3 Inflammasome Assembly.

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1ß (IL-1ß) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1ß maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1ß than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1ß secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1ß activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1ß secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1ß activation.IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases.

Infection with a respiratory virus before hematopoietic cell transplantation is associated with alloimmune-mediated lung syndromes.

BACKGROUND: Alloimmune-mediated lung syndromes (allo-LSs) are life-threatening complications after hematopoietic cell transplantation (HCT). Respiratory virus (RV) has been suggested to play a role in the pathogenesis. OBJECTIVE: We studied the relation between RV DNA/RNA detection in the upper/lower airways before HCT and the occurrence of allo-LSs. METHODS: We retrospectively analyzed all HCT recipients between 2004 and 2014, in whom real-time PCR for RV was performed in nasopharyngeal aspirates (NPAs) and bronchoalveolar lavage (BAL) fluid before HCT. The main outcome of interest was the presence of an allo-LS, which was defined as idiopathic pneumonia syndrome or bronchiolitis obliterans syndrome. Other outcomes were overall survival and treatment-related mortality. We used Cox proportional hazard models, logistic regression models, and Fine-Gray competing risk regression for analyses. RESULTS: One hundred seventy-nine children (median age, 6.8 years) were included. RVs were found in 61% (41% in BAL fluid/NPAs and 20% in NPAs only). Rhinovirus was the most frequently detected RV (42%). Allo-LSs occurred in 13%. RV positivity in BAL fluid was a predictor for allo-LSs (hazard ratio, 3.8; 95% CI, 1.4-10.7; P = .01), whereas RV positivity in NPAs only was not. No other predictors were found. Grade II to IV acute graft-versus-host disease related to steroid treatment shows a trend toward a protective effect (odds ratio, 0.16; 95% CI, 0.0-1.3; P = .08). Allo-LSs significantly increased treatment-related mortality (52% ± 10% in allo-LSs and 20% ± 4% in non-allo-LSs, P = .007). CONCLUSIONS: These results show that pre-HCT BAL fluid RV positivity was a predictor for allo-LSs. Screening for RVs before HCT might identify patients at risk for allo-LSs. This could have implications for prevention and treatment and might subsequently influence the outcomes of HCT.

Tissue-specific programming of memory CD8 T cell subsets impacts protection against lethal respiratory virus infection.

How tissue-specific anatomical distribution and phenotypic specialization are linked to protective efficacy of memory T cells against reinfection is unclear. Here, we show that lung environmental cues program recently recruited central-like memory cells with migratory potentials for their tissue-specific functions during lethal respiratory virus infection. After entering the lung, some central-like cells retain their original CD27hiCXCR3hi phenotype, enabling them to localize near the infected bronchiolar epithelium and airway lumen to function as the first line of defense against pathogen encounter. Others, in response to local cytokine triggers, undergo a secondary program of differentiation that leads to the loss of CXCR3, migration arrest, and clustering within peribronchoarterial areas and in interalveolar septa. Here, the immune system adapts its response to prevent systemic viral dissemination and mortality. These results reveal the striking and unexpected spatial organization of central- versus effector-like memory cells within the lung and how cooperation between these two subsets contributes to host defense.

Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but not Neutrophils, Promote Viral Clearance after Therapy with Cyclophosphamide and Dexamethasone.

In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus-specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential.

Sendai Virus Induces Persistent Olfactory Dysfunction in a Murine Model of PVOD via Effects on Apoptosis, Cell Proliferation, and Response to Odorants.

BACKGROUND: Viral infection is a common cause of olfactory dysfunction. The complexities of studying post-viral olfactory loss in humans have impaired further progress in understanding the underlying mechanism. Recently, evidence from clinical studies has implicated Parainfluenza virus 3 as a causal agent. An animal model of post viral olfactory disorders (PVOD) would allow better understanding of disease pathogenesis and represent a major advance in the field. OBJECTIVE: To develop a mouse model of PVOD by evaluating the effects of Sendai virus (SeV), the murine counterpart of Parainfluenza virus, on olfactory function and regenerative ability of the olfactory epithelium. METHODS: C57BL/6 mice (6-8 months old) were inoculated intranasally with SeV or ultraviolet (UV)-inactivated virus (UV-SeV). On days 3, 10, 15, 30 and 60 post-infection, olfactory epithelium was harvested and analyzed by histopathology and immunohistochemical detection of S-phase nuclei. We also measured apoptosis by TUNEL assay and viral load by real-time PCR. The buried food test (BFT) was used to measure olfactory function of mice at day 60. In parallel, cultured murine olfactory sensory neurons (OSNs) infected with SeV or UV-SeV were tested for odorant-mixture response by measuring changes in intracellular calcium concentrations indicated by fura-4 AM assay. RESULTS: Mice infected with SeV suffered from olfactory dysfunction, peaking on day 15, with no loss observed with UV-SeV. At 60 days, four out of 12 mice infected with SeV still had not recovered, with continued normal function in controls. Viral copies of SeV persisted in both the olfactory epithelium (OE) and the olfactory bulb (OB) for at least 60 days. At day 10 and after, both unit length labeling index (ULLI) of apoptosis and ULLI of proliferation in the SeV group was markedly less than the UV-SeV group. In primary cultured OSNs infected by SeV, the percentage of cells responding to mixed odors was markedly lower in the SeV group compared to UV-SeV (P = 0.007). CONCLUSION: We demonstrate that SeV impairs olfaction, persists in OE and OB tissue, reduces their regenerative ability, and impairs the normal physiological function of OSNs without gross cytopathology. This mouse model shares key features of human post-viral olfactory loss, supporting its future use in studies of PVOD. Further testing and development of this model should allow us to clarify the pathophysiology of PVOD.

Phosphatase PP4 Negatively Regulates Type I IFN Production and Antiviral Innate Immunity by Dephosphorylating and Deactivating TBK1.

The effective recognition of viral infection and subsequent type I IFN production is essential for the host antiviral innate immune responses. The phosphorylation and activation of kinase TANK-binding kinase 1 (TBK1) plays crucial roles in the production of type I IFN mediated by TLR and retinoic acid-inducible gene I-like receptors. Type I IFN expression must be tightly regulated to prevent the development of immunopathological disorders. However, how the activated TBK1 is negatively regulated by phosphatases remains poorly understood. In this study, we identified a previously unknown role of protein phosphatase (PP)4 by acting as a TBK1 phosphatase. PP4 expression was upregulated in macrophages infected with RNA virus, vesicular stomatitis virus, and Sendai virus in vitro and in vivo. Knockdown of PP4C, the catalytic subunit of PP4, significantly increased type I IFN production in macrophages and dentritic cells triggered by TLR3/4 ligands, vesicular stomatitis virus, and Sendai virus, and thus inhibited virus replication. Similar results were also found in peritoneal macrophages with PP4C silencing in vivo and i.p. infection of RNA virus. Accordingly, ectopic expression of PP4C inhibited virus-induced type I IFN production and promoted virus replication. However, overexpression of a phosphatase-dead PP4C mutant abolished the inhibitory effects of wild-type PP4C on type I IFN production. Mechanistically, PP4 directly bound TBK1 upon virus infection, then dephosphorylated TBK1 at Ser(172) and inhibited TBK1 activation, and subsequently restrained IFN regulatory factor 3 activation, resulting in suppressed production of type I IFN and IFN-stimulated genes. Thus, serine/threonine phosphatase PP4 functions as a novel feedback negative regulator of RNA virus-triggered innate immunity.

[THE VALUE OF RECURRENT BRONCHIAL OBSTRUCTION ASSOCIATED WITH RESPIRATORY INFECTIONS IN INFANTS].

It is shown that recurrent bronchial obstruction with nonallergic genesis is a significant problem for young children. Further researches of this disease are needed in order to develop differentiated therapies.

TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infection (49 d after inoculation). At this time, IL-13 and the adapter protein DAP12 promote TREM-2 cleavage to sTREM-2 that is unexpectedly active in preventing macrophage apoptosis. The results thereby define an unprecedented mechanism for a feed-forward expansion of lung macrophages (with IL-13 production and consequent M2 differentiation) that further explains how acute infection leads to chronic inflammatory disease.

Structure-function analysis of CCL28 in the development of post-viral asthma.

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded ß-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.

Impaired CD8(+) T cell immunity after allogeneic bone marrow transplantation leads to persistent and severe respiratory viral infection.

RATIONALE: Bone marrow transplant (BMT) recipients experience frequent and severe respiratory viral infections (RVIs). However, the immunological mechanisms predisposing to RVIs are uncertain. Therefore, we hypothesized that antiviral T cell immunity is impaired as a consequence of allogeneic BMT, independent of pharmacologic immunosuppression, and is responsible for increased susceptibility to RVI. METHODS: Bone marrow and splenocytes from C57BL/6(H2(b)) mice were transplanted into B10.BR(H2(k)) (Allo) or C57BL/6(H2(b)) (Syn) recipients. Five weeks after transplantation, recipient mice were inoculated intranasally with mouse parainfluenza virus type 1 (mPIV-1), commonly known as Sendai virus (SeV), and monitored for relevant immunological and disease endpoints. MAIN RESULTS: Severe and persistent airway inflammation, epithelial injury, and enhanced mortality are found after viral infection in Allo mice but not in control Syn and non-transplanted mice. In addition, viral clearance is delayed in Allo mice as evidenced by prolonged detection of viral transcripts at Day 15 post-inoculation (p.i.) but not in control mice. In concert with these events, we also detected decreased levels of total and virus-specific CD8(+) T cells, as well as increased T cellexpression of inhibitory receptor programmed death-1 (PD-1), in the lungs of Allo mice at Day 8 p.i. Adoptive transfer of CD8(+) T cells from non-transplanted mice recovered from SeV infection into Allo mice at Day 8 p.i. restored normal levels of viral clearance, epithelial repair, and lung inflammation. CONCLUSIONS: Taken together these results indicate that allogeneic BMT results in more severe RVI based on the failure to develop an appropriate pulmonary CD8(+) T cell response, providing an important potential mechanism to target in improving outcomes of RVI after BMT.