Alveolar inflammation, elevated interleukin-17A (IL-17A), and fibrin deposition are common features in all forms of lung injury followed by fibrotic repair. Type II alveolar epithelial cell (AEC) viability, regulated by tumor suppressor protein p53 and changes in uPA-mediated fibrinolysis, has been linked to lung injury and pulmonary fibrosis (PF). Nevertheless, mechanistic details linking increased IL-17A with p53 and PAI-1 to lung injury and remodeling remain unclear. We found that IL-17A and its receptor (IL-17RA) are induced during various lung injuries. IL-17A augments IL-17RA, p53 and downstream PAI-1 with a concurrent decrease in uPA and its receptor (uPAR) in AECs. These changes promote AEC apoptosis, alveolar injury and PF. In addition, IL-17A causes a dose-dependent increase in IL-17RA and profibrogenic markers in lung fibroblasts (LFs), suggesting myofibroblast differentiation. We further found that inhibition of IL-17A by caveolin-1 scaffolding domain peptide (CSP) or its 7-mer deletion fragment (CSP7) inhibits AEC apoptosis, lung inflammation, and profibrogenic markers in LFs and PF. Further, treatment of mice with bleomycin-induced lung injury using CSP7, an anti-IL-17A antibody, or an IL-17RA blocking antibody attenuates total lung hydroxyproline and soluble collagen content, as well as levels of profibrogenic markers. These observations support the role of IL-17A/IL-17RA signaling in lung injury and post-injury remodeling.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis ( Mtb ) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb -infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Abstract Purpose: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers. Experimental Design: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development. Results: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis–associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors. Conclusions: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348–60. ©2016 AACR.

SH3GL2 depletion in BCa cells.

Association between SH3GL2/MFN2 loss and progression.

SH3GL2 introduction alters various growth regulatory molecules.

Influence of SH3GL2 overexpression in BCa cells.

AbstractThe continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistantMycobacterium tuberculosis(Mtb) infections. In this study, we tested three bacteriophage strains for theirMtb-killing activities and found that two of them efficiently lysedMtbH37Rv in 7H10 agar plates. However, only phage DS6A efficiently killed H37Rv in liquid culture and inMtb-infected human primary macrophages. In subsequent experiments, we infected humanized mice with aerosolized H37Rv, then treated these mice with DS6A intravenously to test itsin vivoefficacy. We found that DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reducedMtbload in mouse organs with greater efficacy in the spleen. These results demonstrated the feasibility of developing phage therapy as an effective therapeutic againstMtbinfection.

Legends for Supplementary Figures 1-6.

Phosphorylation sites in SH3GL2.

Impact of SH3GL2 overexpression in vivo.

Neutrophil infiltration of the lungs is associated with granuloma formation and the severity of tuberculosis infection. Although several cytokines and chemokines are known to contribute to lung neutrophil infiltration, the neutrophilic chemotactic factors of Mycobacterium tuberculosis ( Mtb ) remain unexplored. Therefore, we performed Transwell based chemotactic assays using neutrophils from human peripheral blood and mouse bone marrow to probe the chemotactic activity of the culture filtrates (CF) of Mtb H37Rv. CF of H37Rv induced chemotaxis of both human and mouse neutrophils, and this was also confirmed with CF of 9 clinical isolates and Erdman strain of Mtb with neutrophil chemotactic activity. Sulfasalazine, an N-formyl-Met-Leu-Phe (fMLF) receptor inhibitor, blocked the chemotaxis of neutrophils induced by CF of Mtb , thus indicating the involvement of the fMLF receptor in Mtb CF induced chemotaxis of neutrophils. Mass spectrometry analysis of CF of H37Rv identified three candidate N-formylated heptapeptides. The chemotactic activity of the identified peptides was confirmed with their synthetic mimetics that they induced neutrophil chemotaxis in a manner dependent on N-terminal formylation. For all formylated peptides and CF of Mtb, the induced Ca 2+ influx in neutrophils was suppressed by sulfasalazine. Thus, we identified novel formylated Mtb peptides with neutrophil chemotactic activity.

Histone deacetylases (HDACs) are critical immune regulators. However, their roles in interleukin-1 beta (IL-1 beta) production remain unclear. By screening 11 zinc-dependent HDACs with chemical inhibitors, we found that HDAC1 inhibitor, 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (DHOB), enhanced IL-1 beta production by macrophage and dendritic cells upon TLR4 stimulation or Mycobacterium tuberculosis infection through IL-1 beta maturation via elevated NLRP3 expression, increased cleaved caspase-1, and enhanced ASC oligomerization. DHOB rescued defective IL-1 beta production by dendritic cells infected with M. tuberculosis with ESAT-6 deletion, a virulence factor shown to activate NLRP3 inflammasome. DHOB increased IL-1 beta production and NLRP3 expression in a tuberculosis mouse model. Although DHOB inhibited HDAC activities of both HDAC1 and HDAC2 by direct bin, g, knockdown of HDAC2, but not HDAC1, increased IL-1 beta production and NERP3 expression in M. tuberculosis-infected macrophages. These data suggest that HDAC2, but not HDAC1, controls IL-1 beta production through NLRP3 inflammasome activation, a mechanism with a significance in chronic inflammatory diseases including tuberculosis.

To determine the mechanisms that mediate resistance to Mycobacterium tuberculosis (M. tuberculosis) infection in household contacts (HHCs) of patients with tuberculosis (TB), we followed 452 latent TB infection-negative (LTBI-) HHCs for 2 years. Those who remained LTBI- throughout the study were identified as nonconverters. At baseline, nonconverters had a higher percentage of CD14+ and CD3-CD56+CD27+CCR7+ memory-like natural killer (NK) cells. Using a whole-transcriptome and metabolomic approach, we identified deoxycorticosterone acetate as a metabolite with elevated concentrations in the plasma of nonconverters, and further studies showed that this metabolite enhanced glycolytic ATP flux in macrophages and restricted M. tuberculosis growth by enhancing antimicrobial peptide production through the expression of the surface receptor sialic acid binding Ig-like lectin-14. Another metabolite, 4-hydroxypyridine, from the plasma of nonconverters significantly enhanced the expansion of memory-like NK cells. Our findings demonstrate that increased levels of specific metabolites can regulate innate resistance against M. tuberculosis infection in HHCs of patients with TB who never develop LTBI or active TB.

Nontuberculous mycobacteria (NTM) infection is common in patients with structural lung damage. To address how NTM infection is established and causes lung damage, we established an NTM mouse model by intranasal inoculation of clinical isolates of M. intracellulare. During the 39-week course of infection, the bacteria persistently grew in the lung and caused progressive granulomatous and fibrotic lung damage with mortality exceeding 50%. Lung neutrophils were significantly increased at 1 week postinfection, reduced at 2 weeks postinfection and increased again at 39 weeks postinfection. IL-17A was increased in the lungs at 1-2 weeks of infection and reduced at 3 weeks postinfection. Depletion of neutrophils during early (0-2 weeks) and late (32-34 weeks) infection had no effect on mortality or lung damage in chronically infected mice. However, neutralization of IL-17A during early infection significantly reduced bacterial burden, fibrotic lung damage, and mortality in chronically infected mice. Since it is known that IL-17A regulates matrix metalloproteinases (MMPs) and that MMPs contribute to the pathogenesis of pulmonary fibrosis, we determined the levels of MMPs in the lungs of M. intracellulare-infected mice. Interestingly, MMP-3 was significantly reduced by anti-IL-17A neutralizing antibody. Moreover, in vitro data showed that exogenous IL-17A exaggerated the production of MMP-3 by lung epithelial cells upon M. intracellulare infection. Collectively, our findings suggest that early IL-17A production precedes and promotes organized pulmonary M. intracellulare infection in mice, at least in part through MMP-3 production.

Background: Histone deacetylases (HDACs) are critical in immune regulation, however their significance in Mycobacterium tuberculosis (Mtb) infection remains unexplored.Methods: We studied the roles of HDAC1 in tuberculosis infection by determining the growth of Mtb in macrophages and mouse model of tuberculosis treated with HDAC1 inhibitor, 4-(dimethylamino)-N-[6(hydroxyamino)-6-oxohexyl]-benzamide (DHOB) or DMSO as a control. The growth of Mtb in macrophages with HDAC1 knockdown by gene editing is also examined. The expression of HDAC1 and activation of STAT1 in Mtb-infected macrophages were determined by western blotting. The changes in acetylated proteins in Mtb infected macrophages with HDAC1 knockdown were determined by mass spectrometry analysis and the cytokines in the mice organs were determined by ELISA.Findings: DHOB rendered macrophages and macrophages with IFN-γ unable to control the growth of Mtb. This was confirmed in a mouse model of Mtb infection treated with DHOB. Mtb infection induced phosphorylation of STAT1 in macrophages and IFN-γ further increased the phosphorylation of STAT1 and this was suppressed by DHOB but not HDAC3 inhibitor RGFP966 through increased lysine acetylation of STAT1. DHOB reduced phosphorylated STAT1 in Mtb infected mouse lungs compared to the control mice despite increased IFN-γ production. HDAC1 knockdown in macrophages significantly increased growth of Mtb over control cells and reduced IFN-γ induced phosphorylation of STAT1, increased acetylation of Beclin1 and reduced p62, major autophagy regulators, and reduced iNOS expression.Interpretation: Our data indicate that HDAC1 is required for protection against tuberculosis infection through regulation of the innate immune cell control of Mtb growth.Funding Statement: This research is supported by the funds from the University of Texas Health Science Center at Tyler.Declaration of Interests: The authors have no financial conflicts of interest.Ethics Approval Statement: All the procedures involving animals were performed in accordance with NIH guidelines and regulations and approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Tyler.Venous blood samples were collected from QuantiFERON TB Gold negative or positive healthy donors with signed informed consents following the protocols approved by the Institutional Review Board of the University of Texas Health Science Center at Tyler.

Abstract Background: Nontuberculous mycobacteria (NTM) causes disseminated disease in patients with immunodeficiency and pulmonary disease in individuals without obvious immunodeficiency. There is no clear information on how NTM pulmonary disease (NTMPD) develops in immunocompetent hosts. This profile study was initiated to gain insight into the immunological factors that predispose persons to pulmonary NTM infections.Methods: Blood samples were obtained from 15 pairs of NTMPD patients and age-matched healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the heat-killed M. avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in PBMCs culture supernatants and plasma using multiplex immunoassays. The mRNA expression of TLR2, P2X7R, TWIK2, THIK2 and TREK1 was measured by quantitative real-time PCR. In mechanistic studies, blood samples were obtained from healthy volunteers who had not been diagnosed or treated for NTM.Results: Interleukin (IL)-1β, IL-18, IL-1α and IL-10 production were significantly reduced in response to MAC in PBMCs of NTMPD patients compared with PBMCs of their healthy household contacts. RANTES was the only chemokine significantly elevated in the plasma of NTMPD patients compared with plasma of their healthy household contacts. TLR2 and TWIK2 expression were impaired in response to MAC in PBMCs of NTMPD patients compared with PBMCs of their healthy household contacts. RANTES had no effect on IL-1β production by macrophages infected with MAC. A TLR2 inhibitor decreased IL-1β, IL-18, IL-1α and IL-10 production by MAC-stimulated PBMCs and monocytes. A TWIK2 inhibitor decreased the production of IL-1β, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes.Conclusions: These findings suggest that impaired TWIK2 results in decreased production of IL-1 family cytokines (IL-1β, IL-18, and IL-1α) in response to MAC, consequentially may increase susceptibility to NTM pulmonary infection.

Nontuberculous mycobacteria (NTM) cause pulmonary disease in individuals without obvious immunodeficiency. This study was initiated to gain insight into the immunological factors that predispose persons to NTM pulmonary disease (NTMPD). Blood was obtained from 15 pairs of NTMPD patients and their healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the Mycobacterium avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in plasma and PBMC culture supernatants using multiplex immunoassays, and gene expression in the PBMCs was determined using real-time PCR. PBMCs from NTMPD patients produced significantly less interleukin-1β (IL-1β), IL-18, IL-1α, and IL-10 than PBMCs from their healthy household contacts in response to MAC. Although plasma RANTES levels were high in NTMPD patients, they had no effect on IL-1β production by macrophages infected with MAC. Toll-like receptor 2 (TLR2) and TWIK2 (a two-pore domain K+ channel) were impaired in response to MAC in PBMCs of NTMPD patients. A TLR2 inhibitor decreased all four cytokines, whereas a two-pore domain K+ channel inhibitor decreased the production of IL-1β, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. A reduced monocyte ratio might contribute to the attenuated production of IL-1 family cytokines by PBMCs of NTMPD patients in response to MAC stimulations. Collectively, our findings suggest that the attenuated IL-1 response may increase susceptibility to NTM pulmonary infection through multiple factors, including impaired expression of the TLR2 and TWIK2 and reduced monocyte ratio. IMPORTANCE Upon MAC stimulation, the production of IL-1 family cytokines and IL-10 by PBMCs of NTMPD patients was attenuated compared with that of healthy household contacts. Upon MAC stimulation, the expression of TLR2 and TWIK2 (one of the two-pore domain K+ channels) was attenuated in PBMCs of NTMPD patients compared with that of healthy household contacts. The production of IL-1 family cytokines by MAC-stimulated PBMCs and MAC-infected monocytes of healthy donors was reduced by a TLR2 inhibitor and two-pore domain K+ channel inhibitor. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. Collectively, our data suggest that defects in the expression of TLR2 and TWIK2 in human PBMCs or monocytes and reduced monocyte ratio are involved in the reduced production of IL-1 family cytokines, and it may increase susceptibility to NTM pulmonary infection.

Abstract Although IL-1b is required for the protection against Mycobacterium tuberculosis (Mtb) infection, its uncontrolled production is associated with chronic inflammation and lung damage in Tuberculosis. Epigenetic effector molecules histone deacetylases (HDACs) play critical roles in tumorigenesis and immune regulation, however their roles in IL-1β production remain unexplored. Initial screening of 11 variants of HDAC with their chemical inhibitors identified that inhibition of HDAC-1 promotes secretion of IL-1β and increases the lysine acetylation of histone H3 by primary macrophages and dendritic cells (DCs) in response to LPS/CD40L, IFN-γ stimulation or Mtb infection without affecting pro-IL-1β and IL-6. Inhibition of NLRP3 or Caspase-1 reversed this effect of HDAC-1 inhibition without affecting cell viability, implying the involvement of NLRP3 inflammasome activation. Consistently, HDAC-1 inhibition further elevated the expression of both mRNA and protein of NLRP3 in macrophages and DCs under stimulation and increased levels of cleaved Caspase-1 and mature IL-1β in the culture supernatants. Mtb infection and stimulation with LPS/CD40L of these cells induced increased expression and phosphorylation of HDAC-1 and increased HDAC-1 activity in cell lysates which was suppressed by HDAC-1 inhibitor. Treatment of low dose aerosol Mtb-infected mice with HDAC-1 inhibitor increased IL-1β in mice lungs without affecting IL-6 production. These suggest that HDAC-1 controls IL-1β maturation via regulation of NLRP3 expression and activation, thus suggesting a novel mechanism for the regulation of IL-1β, a major inflammatory cytokine in tuberculosis infection and other inflammatory diseases.