The division of labor between pulmonary phagocytic subsets [macrophage/monocyte and dendritic cell (DC) subpopulations] has been described at the functional level. However, whether these lung phagocytes also display unique spatial distribution remains unclear. Here, to analyze cellular distribution in lung compartments and contacts between phagocyte subpopulations, we established an immunohistochemistry (IHC)-based method to clearly identify murine lung phagocyte subsets in situ based on differential expression of CD11c, CD11b, MHC-II, Langerin and mPDCA-1. Furthermore, we investigated subset-specific functional differences in antigen uptake and spatial changes upon allergic sensitization. Our staining allowed the distinction between alveolar macrophages (AMs), interstitial macrophage (IM) subpopulations, CD11b+ DC subpopulations, CD103+ DCs, and plasmacytoid DCs (pDCs). We identified interstitial regions between airways and around airways as regions of IM/CD11b+ DC/CD103+ DC clusters, where a subset of IMs (IM2) and CD103+ DCs formed intense contacts that decreased upon allergic sensitization. These data indicate functional interactions between both cell types either in steady state or after antigen encounter affecting the development of allergies or tolerance. Furthermore, we observed major antigen uptake in AMs and IMs rather than DC subpopulations that was not restricted to airways and adjacent areas. This will enable to focus future studies to immunologically relevant cellular interactions and to unravel which cells are tipping the balance between pro-inflammatory immune responses or tolerance.

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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 serine peptidase inhibitor kazal-type (SPINK)7 is down-regulated in eosinophilic esophagitis (EoE), a type-2 immune disease of the esophagus. Loss of SPINK7 in esophageal epithelial cells unleashes a pro-Inflammatory response and loss of barrier function. Herein, we investigated the mechanism by which SPINK7 mediates its function. METHODS: We performed SPINK7 silencing and genetic depletion of SPINK7 in human esophageal epithelial cells that were cultured at the airliquid interface. Protease activity of SPINK7 deficient epithelial cells and human esophageal tissue was measured, and receptor expression of esophageal tissue-derived eosinophils was quantified. We assessed whether genetic variants in atopic genes were associated with EoE susceptibility. In vitro assays using recombinant proteins were conducted to identify direct targets of SPINK7. Last, we used murine-EoE model to identify the role of SPINK7 target protease; Kallikrein (KLK)5. RESULTS: Epithelial deficiency of SPINK7 promoted increased urokinase plasminogen activator (uPA) and trypsin-like activities (1.8-fold and 3-fold; p50.013, p50.004 respectively). uPA activity was increased by 10-fold in the esophagus of EoE patients as compared to control individuals (p50.043). uPA had the capacity to promote eosinophil activation. Genetic studies revealed a strong epistasis between genetic variants in PLAU (gene product, uPA) with the atopy risk variant in TSLP gene. Furthermore, we revealed KLK5 as SPINK7 target (K i 5132 nM). KLK5 deficient mice had 5-fold decrease in eosinophil infiltration to the esophagus as compared to wt mice (p50.0008). CONCLUSIONS: We propose that loss of SPINK7 in the esophagus unleashes uncontrolled activity of KLK5 and uPA which regulate eosinophil infiltration and activation in the esophagus.