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Liver-dependent lung remodeling in the wake of systemic inflammation shapes responses to secondary infection

ABSTRACT: :Systemic duress, like that elicited by sepsis, burns or trauma, predispose patients to nosocomial pneumonia, demanding better understanding of host pathways influencing this deleterious connection. These pre-existing circumstances are capable of triggering the hepatic acute phase response (APR), which we previously demonstrated is essential for limiting susceptibility to secondary lung infections. To identify potential mechanisms underlying protection afforded by the lung-liver axis, our studies aimed to evaluate liver-dependent lung reprogramming when a systemic inflammatory challenge precedes pneumonia. WT mice and APR-deficient littermates lacking hepatocyte STAT3 (hepSTAT3-/-), a transcription factor necessary for full APR initiation, were challenged intraperitoneally with LPS to induce endotoxemia. After 18h, pneumonia was induced by intratracheal E. coli instillation. Endotoxemia elicited significant transcriptional alterations in the lungs of WT and hepSTAT3-/- mice, with nearly 2,000 differentially expressed genes between genotypes. The gene signatures revealed exaggerated immune activity in the lungs of hepSTAT3-/- mice, which were compromised in their capacity to launch additional cytokine responses to secondary infection. Proteomics revealed substantial liver-dependent modifications in the airspaces of pneumonic mice, implicating a network of dispatched liver-derived mediators influencing lung homeostasis. These results indicate that following systemic inflammation, liver acute phase changes dramatically remodel the lungs, resulting in a modified landscape for any stimuli encountered thereafter. Based on the established vulnerability of hepSTAT3-/- mice to secondary lung infections, we believe that intact liver function is critical for maintaining the immunological responsiveness of the lungs. Further studies are needed to confirm whether and how such lung changes directly influence pneumonia susceptibility.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Bronchoalveolar Lavage

DISEASE(S): Pneumonia

SUBMITTER: TuKiet Lam

LAB HEAD: TuKiet Lam

PROVIDER: PXD024663 | Pride | 2021-07-23

REPOSITORIES: Pride

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Project description:Systemic duress such as that elicited by sepsis, burns or trauma predispose patients to nosocomial pneumonia, demanding a better understanding of host pathways influencing this connection. These systemic challenges are also capable of triggering the hepatic acute phase response (APR), an event that we have previously demonstrated as essential for limiting susceptibility to secondary lung infections. In an effort to identify potential mechanisms underlying protection afforded by the lung-liver axis, our current studies aimed to comprehensively evaluate liver-dependent lung reprogramming following a systemic inflammatory challenge with endotoxemia followed by pneumonia. To do so, WT mice and APR-deficient littermates lacking hepatocyte STAT3 (hepSTAT3-/-), a transcription factor necessary for full APR initiation, were challenged intraperitoneally with LPS to induce endotoxemia. After 18h, pneumonia was induced by an intratracheal E. coli instillation. Lung transcriptional profiling, airspace proteomics, and additional validation measures were assessed after endotoxemia with or without a subsequent challenge with pneumonia in order to achieve an unbiased assessment of lung immune activity in the presence and absence of an intact liver response. In WT mice, endotoxemia elicited robust transcriptional changes in the lungs, with nearly 2,000 differentially expressed genes when comparing WT and hepSTAT3-/- mice. The resulting gene signatures revealed highly exaggerated immune activity in the lungs of hepSTAT3-/- mice, which were compromised in their capacity to launch additional cytokine responses to secondary infection. Proteomics also revealed substantial liver-dependent modifications in the airspaces of pneumonic mice, implicating a network of dispatched liver-derived mediators influencing lung homeostasis. These results indicate that following a systemic inflammatory event, liver acute phase changes dramatically remodel the lungs, resulting in a modified landscape for any stimuli encountered thereafter. Based on the known vulnerability of hepSTAT3-/- mice to secondary lung infections, we believe that intact liver function is critical for maintaining the immunological responsiveness of the lungs. However, further studies are needed to confirm whether and how such lung changes directly influence pneumonia susceptibility.

Project description:Vaccination reduces morbidity and mortality from pneumonia but its effect on the tissue-level response to infection is still poorly understood. We evaluated pneumonia disease progression, acute phase response and lung gene expression profiles in mice inoculated intranasally with virulent gram-positive Streptococcus pneumoniae serotype (ST) 3, with and without prior immunization with pneumococcal polysaccharide ST 3 (PPS3), or co-immunization with PPS3 and with a low dose of lipopolysaccharide (LPS). Pneumonia severity was assessed in the acute phase, 5, 12, 24 and 48 h post-inoculation (p.i.) and the resolution phase of 7 days p.i. Primary PPS3 specific antibody production was upregulated and IgM binding to pneumococci increased in PPS3-immunized mice. Immunizations with PPS3 or PPS3 + LPS decreased bacterial recovery the lung and blood at 24 and 48 h and increased survival. Microarray analysis of whole lung RNA revealed significant changes in the acute phase protein serum amyloid A (SAA) between noninfected and infected mice, which were attenuated by immunization. SAA transcripts were higher in the liver and lungs of infected controls, and SAA protein was elevated in serum, but decreased in PPS3-immunized mice. Thus, during a virulent pneumonia infection, prior immunization with PPS3 in an IgM-dependent manner as well as co-immunization with PPS3 + LPS attenuated pneumonia severity and promoted resolution of infection, concomitant with significant regulation of cytokine gene expression in the lungs, and acute phase proteins in the lungs, liver and serum. Each lung RNA sample represented an individual mouse, creating biological repeats for each treatment. In-vivo treatments were as follows: non-infected lung (vehicle-immunized) control (n=5), infected lung (vehicle-immunized) control at 48 hr post-inoculation (n=5), PPS3 and LPS co-immunized lung at 48 hr post-inoculation (n=4) and PPS3 and LPS co-immunized lung at 7 days post-inoculation (n=4).

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.

Project description:Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is an inflammatory process of the lungs characterized by increased permeability of the alveolar-capillary membrane with subsequent interstitial/alveolar edema and diffuse alveolar damage. ALI/ARDS can be the results of either direct or indirect lung injury, with pneumonia being the most common direct pulmonary insult and sepsis the most common extra-pulmonary cause. In this study, we employed the murine lipopolysaccharide (LPS)-induced direct and indirect lung injury model to explore the pathogenic mechanisms of pulmonary and extra-pulmonary ARDS, using an unbiased, discovery and quantitative proteomic approach. A total of 1,017 proteins were both identified and quantified in bronchoalveolar lavage fluid (BALF) from control, intratracheal LPS (I.T. LPS, 0.1 mg/kg) and intraperitoneal LPS (I.P. LPS, 5 mg/kg) treated mice. The two LPS groups shared 13 up-regulated and 22 down-regulated proteins compared to the control group. Among them, molecules related to bronchial and type II alveolar epithelial cell functions including cell adhesion molecule 1 and surfactant protein B were reduced, whereas lactotransferrin and resistin like alpha involved in lung innate immunity were upregulated in both LPS groups. Proteomic profiling also identified significant differences in BALF proteins between I.T. and I.P. LPS groups. Ingenuity pathway analysis revealed that acute-phase response signaling was activated by both I.T. and I.P. LPS, however, the magnitude of activation is much greater in I.T. LPS group compared to I.P. LPS group. Intriguingly, two canonical signaling pathways, liver X receptor/retinoid X receptor activation and the production of nitric oxide and reactive oxygen species in macrophages, were activated by I.T. LPS but suppressed by I.P. LPS. In addition, CXCL15 (also known as lungkine) was also up-regulated by I.T LPS but down-regulated by I.P. LPS. In conclusion, our quantitative discovery-based proteomic approach identified commonalities as well as significant differences in BALF protein expression profiles in LPS-induced direct and indirect lung injury, and importantly, LPS-induced indirect lung injury results in suppression of select components of lung innate immunity, which could contribute to the so-called “immunoparalysis” in sepsis patients.

Project description:Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and Class A bioterror bacterial pathogens, and Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-kappaB, Type I and II interferon, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly upregulated. Taken together, stimulated innate resistance (StIR) appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection. Keywords: differential gene expression; time course; innate immunity; pneumonia; immunocompromised host; lung epithelium Gene expression patterns in mouse lung homogenates were analyzed 2h after exposure to aerosolized PBS (Sham treatment), 2h after exposure to aerosolized NTHi lysate or 4h after exposure to aerosolized NTHi lysate. Each group consisted of six mice.

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Liver-dependent lung remodeling in the wake of systemic inflammation shapes responses to secondary infection

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