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Influenza A virus (IAV) is one of the most ubiquitous respiratory viruses in the world, causing significant disease burden in the United States and abroad. Current vaccination strategies that target the generation of humoral immunity offer limited heterosubtypic protection; T cells offer cross-strain protection and the promise of universal immunity against IAV. Local tissue immunity plays a key role in pathogen clearance and tissue protection, particularly in the form of tissue resident memory T cells (TRM), which are a non-circulating memory T cell subset that have been shown in a variety of tissue sites to be superior mediators of protection compared to circulating memory T cells. At the same time, T cell immunity has been associated with inflammatory processes that may also lead to lung immunopathology. How lung tissue localized T cell immunity mediates its protection during a recall response to IAV challenge is not well understood. Using the lymphocyte sequestering drug FTY720, we show that primary infection with H3N2 IAV strain X31 provides tissue localized heterosubtypic immunity independently of humoral immunity against an H1N1 PR8 IAV strain. Within the lung resident niche, the recall response drives faster CD4+ and CD8+ T cell expansion compared to a primary infection. This rapid T cell expansion resulted from in situ TRM proliferation that was augmented by the migration of peripheral T cells. By tracking a naïve T cell population specific for the IAV strain used in secondary challenge, we demonstrate that influenza-specific T cells, including those specific for newly introduced antigens, migrate to the lung niche from the local mediastinal lymph node (medLN) where both CD4+ and CD8+ T cells experience enhanced priming and proliferation. We further show that primary infection fortifies the medLN with persistently increased numbers of T cells as well as both CD103+ and CD103- conventional dendritic cells (cDCs) that are transcriptionally similar to cDCs in an infection naïve mouse. By depleting Zbtb46+ cDCs, we determine that cDC fortification is a crucial mechanism for enhanced T cell priming and expansion in the medLN during a recall response. We also found that lung localized CD4+ T cell responses exhibit significant immunomodulatory function. Polyclonal lung CD4+ TRM generated by influenza infection as well as lung OT-II TRM exhibit increased production of antiviral inflammatory cytokines in addition to enhanced IL-10 family cytokine production compared to splenic CD4+ effector memory T cells (TEM). During a heterosubtypic challenge, we further observed that lung niche non-TRM CD4+ T cells produce significantly more in situ IL-10 compared to a primary infection, which modulated airway IFN-ɣ and TNF-α production without any depreciation in viral clearance. Immunomodulatory characteristics of a recall response was reflected in lung tissue-wide transcriptional downregulation of innate responses such as type I IFN responses compared to a primary infection. This work demonstrates the dual antiviral and immunomodulatory protective role of enhanced tissue-localized T cell responses during the recall response to IAV challenge.

The hematopoietic system has long been charactered for its essential function in protecting against pathogens, but it is increasingly established that immune cells play integral roles in resolving inflammation and driving tissue repair. While many cell types are recruited to the site of injury and participate in coordinated immune responses, regulatory T (Treg) cells have emerged as key players of tissue protection by limiting damage and promoting regeneration in multiple organ systems. A conserved feature of “pro-repair” Treg cells is their expression of amphiregulin (Areg), an epidermal growth factor (EGFR) ligand associated with many formative processes in organismal development, tissue regeneration, and cancer. Many hematopoietic and non-hematopoietic cells produce Areg, yet Treg–specific expression has been found to be uniquely important and non-redundant in a number of damage models such as ischemic stroke, muscle injury, and influenza infection. In the lung, re-establishing epithelial barrier integrity is essential for recovery after acute viral injury. Rapid activation of renewal pathways preserves respiratory function during active inflammation and prevents against secondary infections and sequela. It has been previously reported that during influenza virus infection, Treg cell-production of Areg supports host resilience and thwarts severe alveolar damage. Animals that genetically lack Areg from Treg sources suffer a sharp loss of blood oxygenation and worse pathology. Although this growth factor signaling heavily influences disease outcome, the mechanisms by which Areg signals and how Treg cells engage with parenchymal and stromal cells within the alveolar niche are poorly understood. Given that Treg cells constitute only a small fraction of Areg-producing cells in the lung, we hypothesized that spatially restricted signaling and local tissue interactions enable this minority population to exert a major impact on organ function. Here, I used a multidisciplinary approach to interrogate the ability of lung Treg cells to promote alveolar lung repair during H1N1 influenza infection in a murine system. Through high-resolution immunofluorescence imaging, I characterize the unique distribution of Treg cells within lung tissues and their rapid recruitment to sites of active viral replication. Treg cells co-localize with a distinct population of Collagen-14+ EGFR+ mesenchymal cells (Col14+) that are Areg–responsive and robustly promote alveolar epithelial cell development. In the absence of Treg–derived Areg, Col14+ cells exhibit aberrant transcriptional programming, reduced expression of important alveolar growth factors, Fgf7 and Fgf10, and a dramatic increase in apoptotic cell death that together results in impaired alveolar epithelial progenitor cell differentiation. Following genetic ablation of stromal Egfr expression, mice experience a stark decline in blood oxygen saturation and dysplastic alveolar repair similar to loss of Areg from Treg cells, providing evidence that Areg from Treg cells instead signals through Col14+ cell intermediates. These findings underscore that localized delivery of distinct growth factors within tissue stem niches profound impacts whole organ physiology and regeneration. Lastly, I developed a novel Areg reporter mouse strain to better understand Areg producing cells in vivo. Through multiplexed, gene expression and TCR single-cell RNA sequencing, I identified the distinct factors and TCR repertoire that distinguishes “pro-repair” Treg cells in both influenza and bleomycin-induced lung injury. This system can be used as a platform for investigating the unique mechanisms by which reparative Treg cells and other Areg-producing immune cells migrate within tissues and deliver context-specific signals that orchestrate regenerative programming.

Orientador: Ana Marisa Fusco Almeida / Coorientador: Paulo César Gomes / Banca: Maria Lúcia Taylor / Banca: Mário Sérgio Palma / Banca: Guilherme Targino Valente / Banca: Daniel Guariz Pinheiro / Resumo: Histoplasma capsulatum variedade capsulatum é um patógeno fúngico dimórfico causador de uma importante micose sistêmica, denominada histoplasmose. A patogenia da histoplasmose ocorre como resultado da inalação dos microconídios da fase miceliar, que afetam primariamente os pulmões onde ocorre a diferenciação em leveduras, que posteriormente, induzem uma infecção pulmonar e disseminação para outros órgãos, particularmente em indivíduos imunocomprometidos. Recentemente, foi estabelecida uma correlação entre o modo de infecção de H. capsulatum e a formação de biofilmes, estruturas caracterizadas como redes tridimensionais complexas que induzem, entre outros, a resistência antifúngica. Por esta razão, é emergente a identificação de um novo alvo e/ou biomarcador que possa ser selecionado, a fim de estabelecer um novo regime terapêutico e/ou ferramenta diagnóstica para a histoplasmose. Com base nisto, este trabalho tem como objetivo analisar a regulação transcricional codificante e metabólica diferencial entre as duas formas de crescimento de H. capsulatum, em biofilmes e em crescimento planctônico, bem como determinar o perfil de microRNAs (miRNAs) aberrantes em células hospedeiras em resposta à infecção com leveduras livres e biofilmes. O sequenciamento completo das regiões transcritas foi realizado utilizando a plataforma HiSeq da Illumina e a investigação do padrão de miRNAs em macrófagos (Mø) hospedeiros infectados foi conduzida empregando uma técnica de RT-qPCR (reverse trans... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Histoplasma capsulatum variety capsulatum is a dimorphic fungal pathogen that causes an important systemic mycosis, called histoplasmosis. The pathogenesis of histoplasmosis occurs as a result of the inhalation of mycelial microconidia, which primarily affect the lungs where differentiation occurs in yeast, which subsequently induce pulmonary infection and dissemination to other organs, particularly in immunocompromised individuals. Recently, a correlation was established between the mode of infection of H. capsulatum and the formation of biofilms, structures characterized as complex three-dimensional networks that induce, among others, antifungal resistance. For this reason, it is emerging the identification of a new target that can be selected in order to establish a new therapeutic strategy for histoplasmosis. Based on this, this work aims to analyze the differential transcriptional and coding transcriptional regulation between the two forms of growth of H. capsulatum in biofilms and planktonic growth, as well as to determine the profile of aberrant microRNAs (miRNAs) in host cells post-infection with free yeasts and biofilms. Complete sequencing of the transcribed regions was performed using the Illumina HiSeq platform and the investigation of the miRNA pattern in infected host macrophages (Mø) was conducted using a reverse transcription-quantitative PCR (RT-qPCR) technique. Subsequently, the production of total polysaccharides was determined in samples of planktonic cultures, biofilms and supernatant of the biofilms and, additionally, the abundance of non-protein metabolites and small peptides in these growth forms was established by LC-MS/MS. In summary, the data obtained showed that the structure of yeasts in biofilms induces a negative regulation in the direct (coding genome - mRNA) and indirectly... (Complete abstract click electronic access below) / Doutor

Pseudomonas aeruginosa is a Gram-negative bacterium implicated in several clinical contexts. In its association with immunocompromised hosts including cystic fibrosis patients, P. aeruginosa is able to exploit the host immune response to acquire key factors essential to its adaptation. As such, key virulence factors including the Type III Secretion System (T3SS), initially essential in acute infection, is reduced in its significance in chronic colonization. On the contrary, other phenotypes are essential for the altered priorities in chronic colonization. The signals of the host immune response initiating the phenotypic switch from the expression of acute virulence factors to chronic virulence factors have not been well defined. Additionally, the function of the type VI secretion system (T6SS), a protein secretion apparatus, in chronic infection has been well established. Clinical isolates obtained from acute and chronic P. aeruginosa infections suggested selective regulation of the T6SS, namely up regulation of the H3-T6SS in chronic infection. We used murine models of infection to understand the in vivo transcriptional regulation of the T6SS of PAO1. Itaconate, an anti-inflammatory metabolite generated by the host, selectively upregulated transcription of a H3-T6SS-associated locus, vgrG3. Here we present evidence to show how the host immune response, namely metabolic changes in response to infection may be exploited to support the organism’s adaptation to the lung microenvironment. In the evaluation of such a phenotypic response notable in chronic infections, the Type VI Secretion System (T6SS) of P. aeruginosa is selectively regulated by a host-specific metabolic product, itaconate. While P. aeruginosa contains genetic clusters for three (H1-, H2-, and H3-T6SS) evolutionarily distinct T6SSs, we found the H3-T6SS to be up-regulated significantly (p<0.05) in the presence of this anti-inflammatory signal. Characterization of this response reveals that itaconate induces metabolic stress in P. aeruginosa. In an acute pneumoniae mouse model, deletion of the H3-T6SS locus results in increased colonization of the murine lung. Analysis of bronchoalveolar lavage fluid from wild type and H3-T6SS null-infected mice reveals alterations in metabolic pathways including purine metabolism, carbon metabolism, and arginine biosynthesis. Overall our work outlines the H3-T6SS as a phenotypic response to metabolic stress induced by the host immune response, serving to mediate pathways essential in pathogenesis. Further understanding of such phenotypes as the T6SS implicated in chronic infection is essential in treatment interventions in the clinic.