isolation-alveolar-type-ii-epithelial-cells-from-neonatal-juvenile

Isolation of Alveolar Type II Epithelial Cells from Neonatal, Juvenile, and Adult Murine Lungs Adaptable to Infectious Experimental Settings

Alveolar type II epithelial cells (AECII) critically contribute to immune regulation in the lung and thus are key elements orchestrating pulmonary ...

Otto-von-Guericke University Magdeburg

Helmholtz Centre for Infection Research

The respiratory tract is constantly exposed to the environment and displays a favorable niche for colonizing microorganisms. However, the effects of respiratory bacterial carriage on the immune system and its implications for secondary responses remain largely unclear. We have employed respiratory carriage with  as the underlying model to comprehensively address effects on subsequent immune responses. Carriage was associated with the stimulation of -specific CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ T cell responses, and broad transcriptional activation was observed in CD4⁺CD25⁺ T cells. Importantly, transfer of leukocytes from carriers to acutely  infected mice, resulted in a significantly increased bacterial burden in the recipient's upper respiratory tract. In contrast, we found that respiratory  carriage resulted in a significant benefit for the host in systemic infection with  Adaptive responses to vaccination and influenza A virus infection, were unaffected by  carriage. These data showed that there were significant immune modulatory processes triggered by  carriage, that differentially affect subsequent immune responses. Therefore, our results demonstrated the complexity of immune regulation induced by respiratory bacterial carriage, which can be beneficial or detrimental to the host, depending on the pathogen and the considered compartment.

Respiratory  Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4⁺CD25⁺ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice.

An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the  strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies.

 Neutralization of Pro-inflammatory Cytokines During Secondary  Infection Post Influenza A Virus Infection.

Transport of secretory immunoglobulin A (SIgA) through the airway epithelial cell barrier into the mucosal lumen by the polymeric immunoglobulin receptor (pIgR) is an important mechanism of respiratory mucosal host defense. Identification of immunomodulating substances that regulate secretory immunity might have therapeutic implications with regard to an improved immune exclusion. Thus, we sought to analyze secretory immunity under homeostatic and immunomodulating conditions in different compartments of the murine upper and lower respiratory tract (URT&LRT).

Exogenous and Endogenous Triggers Differentially Stimulate Pigr Expression and Antibacterial Secretory Immunity in the Murine Respiratory Tract.

The tracking of pathogen burden and host responses with minimally invasive methods during respiratory infections is central for monitoring disease development and guiding treatment decisions. Utilizing a standardized murine model of respiratory influenza A virus (IAV) infection, we developed and tested different supervised machine learning models to predict viral burden and immune response markers, i.e., cytokines and leukocytes in the lung, from hematological data. We performed independently  infection experiments to acquire extensive data for training and testing of the models. We show here that lung viral load, neutrophil counts, cytokines (such as gamma interferon [IFN-γ] and interleukin 6 [IL-6]), and other lung infection markers can be predicted from hematological data. Furthermore, feature analysis of the models showed that blood granulocytes and platelets play a crucial role in prediction and are highly involved in the immune response against IAV. The proposed  tools pave the path toward improved tracking and monitoring of influenza virus infections and possibly other respiratory infections based on minimally invasively obtained hematological parameters.  During the course of respiratory infections such as influenza, we do have a very limited view of immunological indicators to objectively and quantitatively evaluate the outcome of a host. Methods for monitoring immunological markers in a host's lungs are invasive and expensive, and some of them are not feasible to perform. Using machine learning algorithms, we show for the first time that minimally invasively acquired hematological parameters can be used to infer lung viral burden, leukocytes, and cytokines following influenza virus infection in mice. The potential of the framework proposed here consists of a new qualitative vision of the disease processes in the lung compartment as a noninvasive tool.

Predicting Influenza A Virus Infection in the Lung from Hematological Data with Machine Learning.

Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP "OP7" that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.

Generation of "OP7 chimera" defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice.

CD4T cells play a central role in orchestrating the immune response in asthma, with dysregulated ion channel profiles and altered metabolic signatures contributing to disease progression and severity. An important classification of asthma is based on the presence of T-helper cell type 2 (Th2) inflammation, dividing patients into Th2-high and Th2-low endotypes. These distinct endotypes have implications for disease severity, treatment response, and prognosis. By elucidating how ion channels and energy metabolism control Th cells in asthma, this review contributes to the pathophysiological understanding and the prospective development of personalized therapeutic treatment strategies for patients suffering from distinct asthma endotypes.

Metabolic and ionic control of T cells in asthma endotypes.

Asthma is one of the most common chronic respiratory diseases and is characterized by airway inflammation, increased mucus production, and structural changes in the airways. Recently, there is increasing evidence that the disease is much more heterogeneous than expected, with several distinct asthma endotypes. Based on the specificity of T cells as the best-known driving force in airway inflammation, bronchial asthma is categorized into T helper cell 2 (Th2) and non-Th2 asthma. The most studied effector cells in Th2 asthma include T cells and eosinophils. In contrast to Th2 asthma, much less is known about the pathophysiology of non-Th2 asthma, which is often associated with treatment resistance. Besides T cells, the interaction of myeloid cells such as monocytes/macrophages and mast cells with the airway epithelium significantly contributes to the pathogenesis of asthma. However, the underlying molecular regulation and particularly the specific relevance of this cellular network in certain asthma endotypes remain to be understood. In this review, we summarize recent findings on the regulation of and complex interplay between epithelial cells and the "nonclassical" innate effector cells mast cells and monocytes/macrophages in Th2 and non-Th2 asthma with the ultimate goal of providing the rationale for future research into targeted therapy regimens.

Article	Year
Respiratory Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4⁺CD25⁺ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious S... International journal of molecular sciences\| PubMed ID: 30200513	2018
Neutralization of Pro-inflammatory Cytokines During Secondary Infection Post Influenza A Virus Infection. Frontiers in immunology\| PubMed ID: 31474978	2019
Exogenous and Endogenous Triggers Differentially Stimulate Pigr Expression and Antibacterial Secretory Immunity in the Murine Respiratory Tract. Lung\| PubMed ID: 34825965	2022
Predicting Influenza A Virus Infection in the Lung from Hematological Data with Machine Learning. mSystems\| PubMed ID: 36346236	2022
Generation of "OP7 chimera" defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice. Scientific reports\| PubMed ID: 38017026	2023
Metabolic and ionic control of T cells in asthma endotypes. American journal of physiology. Cell physiology\| PubMed ID: 39374078	2024
Innate players in Th2 and non-Th2 asthma: emerging roles for the epithelial cell, mast cell, and monocyte/macrophage network. American journal of physiology. Cell physiology\| PubMed ID: 39401422	2024

Julia D. Boehme

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