C5aR1 activation in mice controls inflammatory eosinophil recruitment and functions in allergic asthma.

BACKGROUND: Pulmonary eosinophils comprise at least two distinct populations of resident eosinophils (rEOS) and inflammatory eosinophils (iEOS), the latter recruited in response to pulmonary inflammation. Here, we determined the impact of complement activation on rEOS and iEOS trafficking and function in two models of pulmonary inflammation. METHODS: BALB/c wild-type and C5ar1-/- mice were exposed to different allergens or IL-33. Eosinophil populations in the airways, lung, or mediastinal lymph nodes (mLN) were characterized by FACS or immunohistochemistry. rEOS and iEOS functions were determined in vivo and in vitro. RESULTS: HDM and IL-33 exposure induced a strong accumulation of iEOS but not rEOS in the airways, lungs, and mLNs. rEOS and iEOS expressed C3/C5 and C5aR1, which were significantly higher in iEOS. Initial pulmonary trafficking of iEOS was markedly reduced in C5ar1-/- mice and associated with less IL-5 production from ILC2 cells. Functionally, adoptively transferred pulmonary iEOS from WT but not from C5ar1-/- mice-induced airway hyperresponsiveness (AHR), which was associated with significantly reduced C5ar1-/- iEOS degranulation. Pulmonary iEOS but not rEOS were frequently associated with T cells in lung tissue. After HDM or IL-33 exposure, iEOS but not rEOS were found in mLNs, which were significantly reduced in C5ar1-/- mice. C5ar1-/- iEOS expressed less costimulatory molecules, associated with a decreased potency to drive antigen-specific T cell proliferation and differentiation into memory T cells. CONCLUSIONS: We uncovered novel roles for C5aR1 in iEOS trafficking and activation, which affects key aspects of allergic inflammation such as AHR, ILC2, and T cell activation.

Delayed Microbial Maturation Durably Exacerbates Th17-driven Asthma in Mice.

Microbial maturation disrupted by early-life dysbiosis has been linked with increased asthma risk and severity; however, the immunological mechanisms underpinning this connection are poorly understood. We sought to understand how delaying microbial maturation drives worsened asthma outcomes later in life and its long-term durability. Drinking water was supplemented with antibiotics on Postnatal Days 10-20. To assess the immediate and long-term effects of delaying microbial maturation on experimental asthma, we initiated house dust mite exposure when bacterial diversity was either at a minimum or had recovered. Airway hyperresponsiveness, histology, pulmonary leukocyte recruitment, flow cytometric analysis of cytokine-producing lymphocytes, and assessment of serum IgG1 (Immunoglobulin G1) and IgE (Immunoglobulin E) concentrations were performed. RT-PCR was used to measure IL-13 (Interleukin 13)-induced gene expression in sequentially sorted mesenchymal, epithelial, endothelial, and leukocyte cell populations from the lung. Delayed microbial maturation increased allergen-driven airway hyperresponsiveness and Th17 frequency compared with allergen-exposed control mice, even when allergen exposure began after bacterial diversity recovered. Blockade of IL-17A (Interleukin 17A) reversed the airway hyperresponsiveness phenotype. In addition, allergen exposure in animals that experienced delayed microbial maturation showed signs of synergistic signaling between IL-13 and IL-17A in the pulmonary mesenchymal compartment. Delaying microbial maturation in neonates promotes the development of more severe asthma by increasing Th17 frequency, even if allergen exposure is initiated weeks after microbial diversity is normalized. In addition, IL-17A-aggravated asthma is associated with increased expression of IL-13-induced genes in mesenchymal, but not epithelial cells.

Modulation of IL-4/IL-13 cytokine signaling in the context of allergic disease.

Aberrant activation of CD4 TH2 cells and excessive production of TH2 cytokines such as IL-4 and IL-13 have been implicated in the pathogenesis of allergic diseases. Generally, IL-4 and IL-13 utilize Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways for induction of inflammatory gene expression and the effector functions associated with disease pathology in many allergic diseases. However, it is increasingly clear that JAK/STAT pathways activated by IL-4/IL-13 can themselves be modulated in the presence of other intracellular signaling programs, thereby changing the overall tone and/or magnitude of IL-4/IL-13 signaling. Apart from direct activation of the canonic JAK/STAT pathways, IL-4 and IL-13 also induce proinflammatory gene expression and effector functions through activation of additional signaling cascades. These alternative signaling cascades contribute to several specific aspects of IL-4/IL-13-associated cellular and molecular responses. A more complete understanding of IL-4/IL-13 signaling pathways, including the precise conditions under which noncanonic signaling pathways are activated, and the impact of these pathways on cellular- and host-level responses, will better allow us to design agents that target specific pathologic outcomes or tailor therapies for the treatment of uncommon disease endotypes.

Prenatal antibiotics exposure does not influence experimental allergic asthma in mice.

Changes in microbiome (dysbiosis) contribute to severity of allergic asthma. Preexisting epidemiological studies in humans correlate perinatal dysbiosis with increased long-term asthma severity. However, these studies cannot discriminate between prenatal and postnatal effects of dysbiosis and suffer from a high variability of dysbiotic causes ranging from antibiotic treatment, delivery by caesarian section to early-life breastfeeding practices. Given that maternal antibiotic exposure in mice increases the risk of newborn bacterial pneumonia in offspring, we hypothesized that prenatal maternal antibiotic-induced dysbiosis induces long-term immunological effects in the offspring that also increase long-term asthma severity. Therefore, dams were exposed to antibiotics (gentamycin, ampicillin, vancomycin) from embryonic day 15 until birth. Six weeks later, asthma was induced in the offspring by repeated applications of house dust mite extract. Airway function, cytokine production, pulmonary cell composition and distribution were assessed. Our study revealed that prenatally induced dysbiosis in mice led to an increase in pulmonary Th17+ non-conventional T cells with limited functional effect on airway resistance, pro-asthmatic Th2/Th17 cytokine production, pulmonary localization and cell-cell contacts. These data indicate that dysbiosis-related immune-modulation with long-term effects on asthma development occurs to a lesser extent prenatally and will allow to focus future studies on more decisive postnatal timeframes.

The balance between protective and pathogenic immune responses to pneumonia in the neonatal lung is enforced by gut microbiota.

Although modern clinical practices such as cesarean sections and perinatal antibiotics have improved infant survival, treatment with broad-spectrum antibiotics alters intestinal microbiota and causes dysbiosis. Infants exposed to perinatal antibiotics have an increased likelihood of life-threatening infections, including pneumonia. Here, we investigated how the gut microbiota sculpt pulmonary immune responses, promoting recovery and resolution of infection in newborn rhesus macaques. Early-life antibiotic exposure interrupted the maturation of intestinal commensal bacteria and disrupted the developmental trajectory of the pulmonary immune system, as assessed by single-cell proteomic and transcriptomic analyses. Early-life antibiotic exposure rendered newborn macaques more susceptible to bacterial pneumonia, concurrent with increases in neutrophil senescence and hyperinflammation, broad inflammatory cytokine signaling, and macrophage dysfunction. This pathogenic reprogramming of pulmonary immunity was further reflected by a hyperinflammatory signature in all pulmonary immune cell subsets coupled with a global loss of tissue-protective, homeostatic pathways in the lungs of dysbiotic newborns. Fecal microbiota transfer was associated with partial correction of the broad immune maladaptations and protection against severe pneumonia. These data demonstrate the importance of intestinal microbiota in programming pulmonary immunity and support the idea that gut microbiota promote the balance between pathways driving tissue repair and inflammatory responses associated with clinical recovery from infection in infants. Our results highlight a potential role for microbial transfer for immune support in these at-risk infants.

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates.

Perinatal inflammatory stress is associated with early life morbidity and lifelong consequences for pulmonary health. Chorioamnionitis, an inflammatory condition affecting the placenta and fluid surrounding the developing fetus, affects 25 to 40% of preterm births. Severe chorioamnionitis with preterm birth is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may markedly alter the development of the lung. Here, we used intra-amniotic lipopolysaccharide (LPS) challenge to induce experimental chorioamnionitis in a prenatal rhesus macaque (Macaca mulatta) model that mirrors structural and temporal aspects of human lung development. Inflammatory injury directly disrupted the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single-cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis that was extensively disrupted by perinatal inflammation, leading to a loss of gas exchange surface and alveolar simplification, with notable resemblance to chronic lung disease in newborns. Blockade of the inflammatory cytokines interleukin-1ß and tumor necrosis factor-α ameliorated LPS-induced inflammatory lung injury by blunting stromal responses to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

A potent myeloid response is rapidly activated in the lungs of premature Rhesus macaques exposed to intra-uterine inflammation.

Up to 40% of preterm births are associated with histological chorioamnionitis (HCA), which leads to elevated levels of pro-inflammatory mediators and microbial products in the amniotic fluid, which come in contact with fetal lungs. Yet, fetal pulmonary immune responses to such exposure remain poorly characterized. To address this gap, we used our established HCA model, in which pregnant Rhesus macaques receive intraamniotic (IA) saline or LPS. IA LPS induced a potent and rapid myeloid cell response in fetal lungs, dominated by neutrophils and monocytes/macrophages. Infiltrating and resident myeloid cells exhibited transcriptional profiles consistent with exposure to TLR ligands, as well as cytokines, notably IL-1 and TNFα. Although simultaneous, in vivo blockade of IL-1 and TNFα signaling did not prevent the inflammatory cell recruitment, it blunted the lung overall inflammatory state reducing communication between, and activation of, infiltrating immune cells. Our data indicate that the fetal innate immune system can mount a rapid multi-faceted pulmonary immune response to in utero exposure to inflammation. These data provide mechanistic insights into the association between HCA and the postnatal lung morbidities of the premature infant and highlight therapeutic potential of inflammatory blockade in the fetus.

Asthma and posttraumatic stress disorder (PTSD): Emerging links, potential models and mechanisms.

Posttraumatic stress disorder (PTSD) is a highly prevalent, debilitating mental health condition. A better understanding of contributory neurobiological mechanisms will lead to effective treatments, improving quality of life for patients. Given that not all trauma-exposed individuals develop PTSD, identification of pre-trauma susceptibility factors that can modulate posttraumatic outcomes is important. Recent clinical evidence supports a strong link between inflammatory conditions and PTSD. A particularly strong association has been reported between asthma and PTSD prevalence and severity. Unlike many other PTSD-comorbid inflammatory conditions, asthma often develops in children, sensitizing them to subsequent posttraumatic pathology throughout their lifetime. Currently, there is a significant need to understand the neurobiology, shared mechanisms, and inflammatory mediators that may contribute to comorbid asthma and PTSD. Here, we provide a translational perspective of asthma and PTSD risk and comorbidity, focusing on clinical associations, relevant rodent paradigms and potential mechanisms that may translate asthma-associated inflammation to PTSD development.

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma.

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.

Pulmonary Consequences of Prenatal Inflammatory Exposures: Clinical Perspective and Review of Basic Immunological Mechanisms.

Chorioamnionitis, a potentially serious inflammatory complication of pregnancy, is associated with the development of an inflammatory milieu within the amniotic fluid surrounding the developing fetus. When chorioamnionitis occurs, the fetal lung finds itself in the unique position of being constantly exposed to the consequent inflammatory meditators and/or microbial products found in the amniotic fluid. This exposure results in significant changes to the fetal lung, such as increased leukocyte infiltration, altered cytokine, and surfactant production, and diminished alveolarization. These alterations can have potentially lasting impacts on lung development and function. However, studies to date have only begun to elucidate the association between such inflammatory exposures and lifelong consequences such as lung dysfunction. In this review, we discuss the pathogenesis of and fetal immune response to chorioamnionitis, detail the consequences of chorioamnionitis exposure on the developing fetal lung, highlighting the various animal models that have contributed to our current understanding and discuss the importance of fetal exposures in regard to the development of chronic respiratory disease. Finally, we focus on the clinical, basic, and therapeutic challenges in fetal inflammatory injury to the lung, and propose next steps and future directions to improve our therapeutic understanding of this important perinatal stress.

Insulin-like Growth Factor 1 Supports a Pulmonary Niche that Promotes Type 3 Innate Lymphoid Cell Development in Newborn Lungs.

Type 3 innate lymphoid cells (ILC3s) are critical for lung defense against bacterial pneumonia in the neonatal period, but the signals that guide pulmonary ILC3 development remain unclear. Here, we demonstrated that pulmonary ILC3s descended from ILC precursors that populated a niche defined by fibroblasts in the developing lung. Alveolar fibroblasts produced insulin-like growth factor 1 (IGF1), which instructed expansion and maturation of pulmonary ILC precursors. Conditional ablation of IGF1 in alveolar fibroblasts or deletion of the IGF-1 receptor from ILC precursors interrupted ILC3 biogenesis and rendered newborn mice susceptible to pneumonia. Premature infants with bronchopulmonary dysplasia, characterized by interrupted postnatal alveolar development and increased morbidity to respiratory infections, had reduced IGF1 concentrations and pulmonary ILC3 numbers. These findings indicate that the newborn period is a critical window in pulmonary immunity development, and disrupted lung development in prematurely born infants may have enduring effects on host resistance to respiratory infections.

Short-term high-fat diet feeding protects from the development of experimental allergic asthma in mice.

BACKGROUND: A close association between obesity and asthma has been described. The nature of this association remains elusive, especially with respect to allergic asthma. Controversial findings exist regarding the impact of short-term high-fat diet (HFD) feeding on the development of allergic asthma. OBJECTIVE: To delineate the impact of short-term HFD feeding on the development of experimental allergic asthma. METHODS: Female C57BL/6JRJ mice were fed with a short-term HFD or chow diet (CD) for 12 weeks. Allergic asthma was induced by intraperitoneal OVA/alum sensitization followed by repeated OVA airway challenges. We determined airway hyperresponsiveness (AHR) and pulmonary inflammation by histologic and flow cytometric analysis of immune cells. Furthermore, we assessed the impact of HFD on dendritic cell (DC)-mediated activation of T cells. RESULTS: Female mice showed a mild increase in body weight accompanied by mild metabolic alterations. Upon OVA challenge, CD-fed mice developed strong AHR and airway inflammation, which were markedly reduced in HFD-fed mice. Mucus production was similar in both treatment groups. OVA-induced increases in DC and CD4+ T-cell recruitment to the lungs were significantly attenuated in HFD-fed mice. MHC-II expression and CD40 expression in pulmonary CD11b+ DCs were markedly lower in HFD-fed compared to CD-fed mice, which was associated in vivo with a decreased T helper (Th) 1/17 differentiation and Treg formation without impacting Th2 differentiation. CONCLUSIONS/CLINICAL RELEVANCE: These findings suggest that short-term HFD feeding attenuates the development of AHR, airway inflammation, pulmonary DC recruitment and MHC-II/CD40 expression leading to diminished Th1/17 but unchanged Th2 differentiation. Thus, short-term HFD feeding and associated metabolic alterations may have protective effects in allergic asthma development.

Immunomodulatory T cell death associated gene-8 (TDAG8) receptor in depression-associated behaviors.

Converging evidence supports neuroimmune factors in depression psychopathology. We previously reported reduced depression-like behavior in immunomodulatory G-protein-coupled receptor, T cell death-associated gene-8 (TDAG8) deficient mice. Here, we expand on those findings by investigating depression- and anxiety-associated behaviors, and cytokine profiles in TDAG8-deficient mice. TDAG8-deficiency reduced depression- and anxiety-associated behaviors in the forced swim test (FST), open-field test and elevated zero maze. Interestingly, cytokine expression, particularly IL-6, was attenuated within hippocampus and spleen in TDAG8-deficient mice following the FST. There were no differences in immune-cell frequencies. Collectively, these data suggest a contributory role of TDAG8 in neuroimmune regulation and depression-associated physiology.

Loss of GTPase of immunity-associated protein 5 (Gimap5) promotes pathogenic CD4+ T-cell development and allergic airway disease.

BACKGROUND: GTPase of immunity-associated protein 5 (GIMAP5) is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 single nucleotide polymorphisms have been linked to an increased risk for asthma, whereas loss of Gimap5 in mice has been associated with severe CD4+ T cell-driven immune pathology. OBJECTIVE: We sought to identify the molecular and cellular mechanisms by which Gimap5 deficiency predisposes to allergic airway disease. METHODS: CD4+ T-cell polarization and development of pathogenic CD4+ T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite-induced airway inflammation was assessed by using a complete Gimap5 LOF (Gimap5sph/sph) and conditional Gimap5fl/flCd4Cre/ert2 mice. RESULTS: GIMAP5 LOF mutations in both mice and human subjects are associated with spontaneous polarization toward pathogenic TH17 and TH2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient TH cell differentiation is associated with increased DNA damage, particularly during TH1-polarizing conditions. DNA damage in Gimap5-deficient CD4+ T cells could be controlled by TGF-ß, thereby promoting TH17 polarization. When challenged with house dust mite in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness), with increased development of TH2, TH17, and pathogenic TH17/TH2 cells. CONCLUSION: Activation of Gimap5-deficient CD4+ T cells is associated with increased DNA damage and reduced survival that can be overcome by TGF-ß. This leads to selective survival of pathogenic TH17 cells but also TH2 cells in human subjects and mice, ultimately promoting allergic airway disease.

Combined administration of anti-IL-13 and anti-IL-17A at individually sub-therapeutic doses limits asthma-like symptoms in a mouse model of Th2/Th17 high asthma.

BACKGROUND: Recent studies have demonstrated that Th2 responses have the ability to antagonize Th17 responses. In mouse models of allergic asthma, blockade of Th2-effector cytokines results in elaboration of Th17 responses and associated increases in pulmonary neutrophilia. While these can be controlled by simultaneous blockade of Th17-associated effector cytokines, clinical trials of anti-IL-17/IL-17RA blocking therapies have demonstrated increased of risk of bacterial and fungal infections. Identification of minimally effective doses of cytokine-blocking therapies with the goal of reducing the potential emergence of infection-related complications is a translationally relevant goal. OBJECTIVE: In the current report, we examine whether combined blockade of IL-13 and IL-17A, at individually sub-therapeutic levels, can limit the development of allergic asthma while sparing expression of IL-17A-associated anti-microbial effectors. METHODS: House dust mite was given intratracheally to A/J mice. Anti-IL-13 and anti-IL-17A antibodies were administered individually, or concomitantly at sub-therapeutic doses. Airway hyper-reactivity, lung inflammation, magnitude of Th2- and Th17-associated cytokine production and expression of IL-13- and IL-17A-induced genes in the lungs was assessed. RESULTS: Initial dosing studies identified sub-therapeutic levels of IL-13 and IL-17A blocking mAbs that have a limited effect on asthma parameters and do not impair responses to microbial products or infection. Subsequent studies demonstrated that combined sub-therapeutic dosing with IL-13 and IL-17A blocking mAbs resulted in significant improvement in airway hyperresponsiveness (AHR) and expression of IL-13-induced gene expression. Importantly, these doses neither exacerbated nor inhibited production of Th17-associated cytokines, or IL-17A-associated gene expression. CONCLUSION: This study suggests that combining blockade of individual Th2 and Th17 effector cytokines, even at individually sub-therapeutic levels, may be sufficient to limit disease development while preserving important anti-microbial pathways. Such a strategy may therefore have reduced potential for adverse events associated with blockade of these pathways.

C3a is required for ILC2 function in allergic airway inflammation.

Aberrant type 2 responses underlie the pathologies in allergic diseases like asthma, yet, our understanding of the mechanisms that drive them remains limited. Recent evidence suggests that dysregulated innate immune factors can perpetuate asthma pathogenesis. In susceptible individuals, allergen exposure triggers the activation of complement, a major arm of innate immunity, leading to the aberrant generation of the C3a anaphylatoxin. C3 and C3a have been shown to be important for the development of Th2 responses, yet remarkably, the mechanisms by which C3a regulates type 2 immunity are relatively unknown. We demonstrate a central role for C3a in driving type 2 innate lymphoid cells (ILC2)-mediated inflammation in response to allergen and IL-33. Our data suggests that ILC2 recruitment is C3a-dependent. Further, we show that ILC2s directly respond to C3a, promoting type 2 responses by specifically: (1) inducing IL-13 and granulocyte-macrophage colony-stimulating factor, whereas inhibiting IL-10 production from ILC2; and (2) enhancing their antigen-presenting capability during ILC-T-cell cross-talk. In summary, we identify a novel mechanism by which C3a can mediate aberrant type 2 responses to aeroallergen exposure, which involves a yet unrecognized cross-talk between two major innate immune components-complement and group 2 innate lymphoid cells.