Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Epithelial immunomodulation by aerosolized Toll-like receptor agonists prevents allergic inflammation in airway mucosa in mice.

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 ("Pam2", TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 ("ODN", TLR9 ligand), when delivered together by aerosol ("Pam2ODN"), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy.

OBIF: an omics-based interaction framework to reveal molecular drivers of synergy.

Bioactive molecule library screening may empirically identify effective combination therapies, but molecular mechanisms underlying favorable drug-drug interactions often remain unclear, precluding further rational design. In the absence of an accepted systems theory to interrogate synergistic responses, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in synergistic biological responses. OBIF performs full factorial analysis of feature expression data from single versus dual exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed transcriptomic and proteomic data of a dyad of immunostimulatory molecules that induces synergistic protection against influenza A and revealed unanticipated NF-κB/AP-1 cooperation that is required for antiviral protection. To demonstrate generalizability, OBIF analyzed data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, unlike existing synergy quantification and prediction methods, OBIF is a phenotype-driven systems model that supports multiplatform interrogation of synergy mechanisms.

Immune Modulation to Improve Survival of Viral Pneumonia in Mice.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8+ T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8+ T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.

Inducible epithelial resistance against acute Sendai virus infection prevents chronic asthma-like lung disease in mice.

BACKGROUND AND PURPOSE: Respiratory viral infections play central roles in the initiation, exacerbation and progression of asthma in humans. An acute paramyxoviral infection in mice can cause a chronic lung disease that resembles human asthma. We sought to determine whether reduction of Sendai virus lung burden in mice by stimulating innate immunity with aerosolized Toll-like receptor (TLR) agonists could attenuate the severity of chronic asthma-like lung disease. EXPERIMENTAL APPROACH: Mice were treated by aerosol with 1-µM oligodeoxynucleotide (ODN) M362, an agonist of the TLR9 homodimer, and 4-µM Pam2CSK4 (Pam2), an agonist of the TLR2/6 heterodimer, within a few days before or after Sendai virus challenge. KEY RESULTS: Treatment with ODN/Pam2 caused ~75% reduction in lung Sendai virus burden 5 days after challenge. The reduction in acute lung virus burden was associated with marked reductions 49 days after viral challenge in eosinophilic and lymphocytic lung inflammation, airway mucous metaplasia, lumenal mucus occlusion and hyperresponsiveness to methacholine. Mechanistically, ODN/Pam2 treatment attenuated the chronic asthma phenotype by suppressing IL-33 production by type 2 pneumocytes, both by reducing the severity of acute infection and by down-regulating Type 2 (allergic) inflammation. CONCLUSION AND IMPLICATIONS: These data suggest that treatment of susceptible human hosts with aerosolized ODN and Pam2 at the time of a respiratory viral infection might attenuate the severity of the acute infection and reduce initiation, exacerbation and progression of asthma.

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.

Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared with day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high-level 4 weeks post challenge. Upregulation of antigen-specific IFN-γ gene expression was observed in rCPAF/CpG-vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared with CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

Guinea pig genital tract lipidome reveals in vivo and in vitro regulation of phosphatidylcholine 16:0/18:1 and contribution to Chlamydia trachomatis serovar D infectivity.

INTRODUCTION: Chlamydia trachomatis (Ct), is the leading cause of sexually transmitted infections worldwide. Host transcriptomic- or proteomic profiling studies have identified key molecules involved in establishment of Ct infection or the generation of anti Ct-immunity. However, the contribution of the host metabolome is not known. OBJECTIVES: The objective of this study was to determine the contribution of host metabolites in genital Ct infection. METHODS: We used high-performance liquid chromatography-mass spectrometry, and mapped lipid profiles in genital swabs obtained from female guinea pigs at days 3, 9, 15, 30 and 65 post Ct serovar D intravaginal infection. RESULTS: Across all time points assessed, 13 distinct lipid species including choline, ethanolamine and glycerol were detected. Amongst these metabolites, phosphatidylcholine (PC) was the predominant phospholipid detected from animals actively shedding bacteria i.e., at 3, 9, and 15 days post infection. However, at days 30 and 65 when the animals had cleared the infection, PC was observed to be decreased compared to previous time points. Mass spectrometry analyses of PC produced in guinea pigs (in vivo) and 104C1 guinea pig cell line (in vitro) revealed distinct PC species following Ct D infection. Amongst these, PC 16:0/18:1 was significantly upregulated following Ct D infection (p < 0.05, >twofold change) in vivo and in vitro infection models investigated in this report. Exogenous addition of PC 16:0/18:1 resulted in significant increase in Ct D in Hela 229 cells. CONCLUSION: This study demonstrates a role for host metabolite, PC 16:0/18:1 in regulating genital Ct infection in vivo and in vitro.

Insights into the development and regulation of T follicular helper cells.

T follicular helper (Tfh) cells are specialized subset of T helper (Th) cells necessary for germinal center reaction, affinity maturation and the differentiation of germinal center B cells to antibody-producing plasma B cells and memory B cells. The differentiation of Tfh cells is a multistage, multifactorial process involving a variety of cytokines, surface molecules and transcription factors. While Tfh cells are critical components of protective immune responses against pathogens, regulation of these cells is crucial to prevent autoimmunity and airway inflammation. Recently, it has been noted that Tfh cells could be potentially implicated either in cancer progression or prevention. Thus, the elucidation of the mechanisms that regulate Tfh cell differentiation, function and fate should highlight potential targets for novel therapeutic approaches. In this review, we summarize the latest advances in our understanding of the regulation of Tfh cell differentiation and their role in health and disease.

T helper 2 and T follicular helper cells: Regulation and function of interleukin-4.

Type 2 immunity is characterized by expression of the cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13, which can function in mediating protective immunity in the host or possess a pathogenic role. T helper (Th) 2 cells have emerged to play a beneficial role in mediating anti-parasitic immunity and are also known to be key players in mediating allergic diseases. In addition to the Th2 cells, recent studies have identified T follicular helper (Tfh) cells as an alternative source of IL-4 to regulate type 2 humoral immune responses, indicating that Th2 and Tfh cells exhibit overlapping phenotypical and functional characteristics. Th2 and Tfh cells appear to utilize distinct mechanisms for regulation of IL-4 expression; however unlike Th2 cells, the regulation and function of Tfh-derived IL-4 is not yet fully understood. Understanding of the molecular mechanisms for IL-4 expression and function in both cell subsets will be beneficial for the development of future therapeutic interventions.

Murine MicroRNA-214 regulates intracellular adhesion molecule (ICAM1) gene expression in genital Chlamydia muridarum infection.

The hallmark of chlamydial infection is the development of upper genital pathology in the form of hydrosalpinx and oviduct and/or tubal dilatation. Although molecular events leading to genital tissue presentation and cellular architectural remodelling are unclear, early-stage host immune responses are believed to contribute to these long-term sequelae. Recently, we reported the contribution of selected infection-associated microRNAs (miRs) in the generation of host immunity at early-stage infection (day 6 after intravaginal Chlamydia muridarum challenge in C57BL/6 mice). In this report, we describe the contribution of an infection-associated microRNA, i.e. miR-214, to host immunity. Chlamydia muridarum infection in the C57BL/6 mouse genital tract significantly down-regulated miR-214 while up-regulating intracellular adhesion molecule 1 (ICAM1) gene expression. These in vivo observations were confirmed by establishing direct regulation of ICAM-1 by miR-214 in ex vivo genital cell cultures in the presence of miR-214 mimic and inhibitor. Because, ICAM-1 contributes to recruitment of neutrophils following infection, we also demonstrated that alteration of ICAM1 by miR-214 in interleukin-17A-deficient (IL-17A(-/-) ) mice correlated with reduction of neutrophils infiltrating genital tissue at day 6 after challenge. Additionally, these early-stage events resulted in significantly decreased genital pathology in IL-17A(-/-) mice compared with C57BL/6 mice. This report provides evidence for early-stage regulation of ICAM1 by microRNAs, resulting in reduction of genital pathology associated with chlamydial infection.

Chlamydia muridarum infection associated host MicroRNAs in the murine genital tract and contribution to generation of host immune response.

PROBLEM: Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection in humans and is associated with reproductive tract damage. However, little is known about the involvement and regulation of microRNAs (miRs) in genital CT. METHODS: We analyzed miRs in the genital tract (GT) following C. muridarum (murine strain of CT) challenge of wild type (WT) and CD4(+) T-cell deficient (CD4(-/-)) C57BL/6 mice at days 6 and 12 post-challenge. RESULTS: At day 6, miRs significantly downregulated in the lower GT were miR-125b-5p, -16, -214, -23b, -135a, -182, -183, -30c, and -30e while -146 and -451 were significantly upregulated, profiles not exhibited at day 12 post-bacterial challenge. Significant differences in miR-125b-5p (+5.06-fold change), -135a (+4.9), -183 (+7.9), and -182 (+3.2) were observed in C. muridarum-infected CD4(-/-) compared to WT mice. In silico prediction and mass spectrometry revealed regulation of miR-135a and -182 and associated proteins, that is, heat-shock protein B1 and alpha-2HS-glycoprotein. CONCLUSION: This study provides evidence on regulation of miRs following genital chlamydial infection suggesting a role in pathogenesis and host immunity.

Use of a Guinea pig-specific transcriptome array for evaluation of protective immunity against genital chlamydial infection following intranasal vaccination in Guinea pigs.

Guinea pigs have been used as a second animal model to validate putative anti-chlamydial vaccine candidates tested in mice. However, the lack of guinea pig-specific reagents has limited the utility of this animal model in Chlamydia sp. vaccine studies. Using a novel guinea pig-specific transcriptome array, we determined correlates of protection in guinea pigs vaccinated with Chlamydia caviae (C. caviae) via the intranasal route, previously reported by us and others to provide robust antigen specific immunity against subsequent intravaginal challenge. C. caviae vaccinated guinea pigs resolved genital infection by day 3 post challenge. In contrast, mock vaccinated animals continued to shed viable Chlamydia up to day 18 post challenge. Importantly, at day 80 post challenge, vaccinated guinea pigs experienced significantly reduced genital pathology - a sequelae of genital chlamydial infections, in comparison to mock vaccinated guinea pigs. Sera from vaccinated guinea pigs displayed antigen specific IgG responses and increased IgG1 and IgG2 titers capable of neutralizing GPIC in vitro. Th1-cellular/inflammatory immune genes and Th2-humoral associated genes were also found to be elevated in vaccinated guinea pigs at day 3 post-challenge and correlated with early clearance of the bacterium. Overall, this study provides the first evidence of guinea pig-specific genes involved in anti-chlamydial vaccination and illustrates the enhancement of the utility of this animal model in chlamydial pathogenesis.

In vivo whole animal body imaging reveals colonization of Chlamydia muridarum to the lower genital tract at early stages of infection.

PURPOSE: The leading cause of sexually transmitted bacterial infection is Chlamydia trachomatis. The aim of this study is to investigate the early events in colonization of this bacterium within the murine genital tract. PROCEDURES: An in vivo animal body imaging technology was used to track fluorophore labeled C. muridarum elementary bodies (EBs) inoculated intravaginally in C57BL/6 mice during the first 24 h of infection. RESULTS: Ascension of viable EBs was observed (1) to be localized to the lower regions of the murine genital tract within the first 24 h post challenge and (2) was dose independent during this early exposure period. Molecular detection revealed enhanced bacterial load in lower regions of the genital tract with increasing bacterial load in the upper region beginning 12 h post inoculation. CONCLUSION: This study provides additional insight into chlamydial colonization in the murine genital tract during the first 12-24 h following inoculation.