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Effect of Batf2 deficiency on gene epxression in murine alveolar macrophages treated ex vivo with IFNg and K. pneumoniae supernatant

ABSTRACT: Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. We previously identified BATF2 among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2-deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage pro-inflammatory gene expression and Kp-induced cytokine responses. In vivo, we observed that Batf2 gene expression was elevated in the lung tissue of wild type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed a modest but significant increase in bacterial burden in the lung, spleen and liver compared to WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with our in vitro observations, pro-inflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances pro-inflammatory cytokine responses in macrophages in response to Kp, and that as such, BATF2 contributes to the host defense against pulmonary Kp infection.

ORGANISM(S): Mus musculus

PROVIDER: GSE235114 | GEO | 2023/08/31

REPOSITORIES: GEO

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Project description:Inflammation is the natural defensive response of the immune system to an injury or infection and is regulated by small molecule mediators triggering different phases of the inflammatory process. In particular, lipid mediators (LM) and cytokines exhibit crucial regulatory functions in the progression and resolution of inflammation. Macrophages play a central role in this process and can adopt distinct phenotypes with specialized functions depending on their microenvironment: inflammatory M1 macrophages drive inflammation by the release of pro-inflammatory cytokines and LMs, like prostaglandins (PG) and leukotrienes (LT), while resolving M2 macrophages promote inflammation resolution and tissue regeneration by production of anti-inflammatory cytokines and specialized pro resolving mediators (SPM). Aging is associated with chronic and unresolved, low-grade inflammation (“inflammaging”) and aging-related dysfunction of macrophages in the resolution of inflammation and tissue maintenance has been reported. Yet, the underlying molecular mechanisms and functional consequences of latter processes remain poorly understood. Here, we show that polarization of peritoneal macrophages (PM) from geriatric mice towards an M2-like phenotype is impaired versus adult mice, resulting in aberrant LM formation and cytokine release. In PMs isolated from adult mice (PM-A) we observed a shift in LM formation from PGs and LTs to SPMs already after 4 h of polarization towards M2 with interleukin (IL) 4. In contrast, PMs from geriatric mice (PM-G) produced mainly LTs and PGs upon polarization. This pattern persists over the course of 48 h of polarization. Proteomic profiling revealed that polarization of PM A towards M2 yields a more distinct phenotype, clearly separated from M1, when compared to PM-G. We observed similar aging-related changes in the lipidome and cytokine profile of spleen, lung and liver tissue from mice. Hence, we hypothesize that during aging macrophage polarization towards M2 is impaired, which in turn drives chronic inflammation and disturbs tissue maintenance. By combining state-of-the art lipidomic and proteomic profiling we aim to uncover new molecular targets for pharmaceutical interventions to improve therapeutic strategies for elderly patients with chronic inflammatory diseases.

Project description:Background: Human lung adenocarcinoma (ADC), the predominant subtype of lung cancer, are at a disadvantage as the disease lacks available biomarkers and is not usually diagnosed until its later stages. We previously demonstrated that Tlr4-mutantmice were more susceptible to BHT-induced pulmonary inflammation (bronchoalveolar lavage macrophages and lymphocytes) and tumorigenesis in comparison to Tlr4-sufficient control mice. Differential transcriptional profiles were also noted in the whole lung tissue of the mice. The study objective was to elucidate the mechanisms underlying the protective effect of Tlr4 including differences in specific innate immune cell populations, their functional responses, and the influence of these cellular differences on the growth of ACD progenitor cell types. Methods: Following butylated hydroxytoluene (BHT) treatment (tumor promoter;4 weekly ip.injections), BALB (Tlr4-sufficient) and C.C3-Tlr4Lps-d/J (BALBLpsd; Tlr4 mutant) lungs were lavaged, processed for flow cytometry, and frozen for molecular analysis (ELISAs for IGF1, KC, Raybiotech Mouse Cytokine Array). BALF-derived macrophages were collected for phagocytosis and efferocytosis assays. BALF macrophage RNA was also isolated and utilized for Affymetrix gene arrays and real-time quantitative PCR. Enrichment of bronchiolar Clara cells, an ADC progenitor cell, was performed and cells enumerated. Femur bone marrow cells were differentiated into macrophages, and co-cultured with C10 cells, a type II cell line, for a wound healing assay. Results: Tlr4-deficiency resulted in significantly increased numbers of innate immune cells in whole lung tissue in response to BHT, including macrophages, PMNs, myeloid-derived suppressor cells (MDSC)s and dendritic cells (DCs), in comparison to Tlr4-sufficient mice (p<05). Macrophage functionality was also affected. BHT treated Tlr4-mutant mice demonstrated enhanced phagocytic and efferocytic abilities and wound healing in comparison to macrophages from Tlr4-sufficient and control treated mice (P<0.05). Additionally, Clara cell proliferation was significantly increased in Tlr4-mutant compared to Tlr4-sufficient mice in response to BHT. Pulmonary cytokine, chemokine and growth factor protein expression also significantly differed among the strains and within macrophages, gene expression and cell surface markers combined demonstrated a more plastic macrophage phenotype in the Tlr4-mutant mice. The transcriptome study identified immune pathways important in inflammation, such as phagosome. Conclusion: There are distinct pulmonary innate immune cell populations, with particular emphasis on macrophages, that are likely influencing the enhanced tumor promotion observed in Tlr4-mutant mice. Though not as striking, the pulmonary lymphoid populations may also be affecting the promotion environment, if only via their influence upon the innate immune cells. Future studies will investigate the origins of the macrophages and continued delineation of the lymphoid cell populations. Two strains of mice were used for these studies. The C.C3-Tlr4Lps-d/J (BALBLpsd) with a dominant negative Tlr4 on a BALB/c background and the BALB/cJ mice with a sufficient Tlr4. Total RNA was extracted from bronchoalveolar lavage fluid macrophages 3 days following a chronic BHT protocol. (oil controls vs BHT treated for both strains. BALB oil and BHT treatment n=3 mice per treatment group; BALBLpsd oil controls n=2 per treatment group; BALBLpsd BHT treated n = 3 per treatment group). Therefore a total of 13 arrays with one BHT exposure times and oil (vehicle) controls.

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Effect of Batf2 deficiency on gene epxression in murine alveolar macrophages treated ex vivo with IFNg and K. pneumoniae supernatant

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