Cell-free hemoglobin triggers macrophage cytokine production via TLR4 and MyD88.

Cell-free hemoglobin (CFH) is elevated in the airspace of patients with acute respiratory distress syndrome (ARDS) and is sufficient to cause acute lung injury in a murine model. However, the pathways through which CFH causes lung injury are not well understood. Toll-like receptor 4 (TLR4) is a mediator of inflammation after detection of damage- and pathogen-associated molecular patterns. We hypothesized that TLR4 signaling mediates the proinflammatory effects of CFH in the airspace. After intratracheal CFH, BALBc mice deficient in TLR4 had reduced inflammatory cell influx into the airspace [bronchoalveolar lavage (BAL) cell counts, median TLR4 knockout (KO): 0.8 × 104/mL [IQR 0.4-1.2 × 104/mL], wild-type (WT): 3.0 × 104/mL [2.2-4.0 × 104/mL], P < 0.001] and attenuated lung permeability (BAL protein, TLR4KO: 289 µg/mL [236-320], WT: 488 µg/mL [422-536], P < 0.001). These mice also had attenuated production of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the airspace. C57Bl/6 mice lacking TLR4 on myeloid cells only (LysM.Cre+/-TLR4fl/fl) had reduced cytokine production in the airspace after CFH, without attenuation of lung permeability. In vitro studies confirm that WT primary murine alveolar macrophages exposed to CFH (0.01-1 mg/mL) had dose-dependent increases in IL-6, IL-1 ß, CXC motif chemokine ligand 1 (CXCL-1), TNF-α, and IL-10 (P < 0.001). Murine MH-S alveolar-like macrophages show TLR4-dependent expression of IL-1ß, IL-6, and CXCL-1 in response to CFH. Primary alveolar macrophages from mice lacking TLR4 adaptor proteins myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-ß (TRIF) revealed that MyD88KO macrophages had 71-96% reduction in CFH-dependent proinflammatory cytokine production (P < 0.001), whereas macrophages from TRIFKO mice had variable changes in cytokine responses. These data demonstrate that myeloid TLR4 signaling through MyD88 is a key regulator of airspace inflammation in response to CFH.NEW & NOTEWORTHY Cell-free hemoglobin (CFH) is elevated in the airspace of most patients with acute respiratory distress syndrome and causes severe inflammation. Here, we identify that CFH contributes to macrophage-induced cytokine production via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling. These data increase our knowledge of the mechanisms through which CFH contributes to lung injury and may inform development of targeted therapeutics to attenuate inflammation.

A two-hit model of sepsis plus hyperoxia causes lung permeability and inflammation.

Mouse models of acute lung injury (ALI) have been instrumental for studies of the biological underpinnings of lung inflammation and permeability, but murine models of sepsis generate minimal lung injury. Our goal was to create a murine sepsis model of ALI that reflects the inflammation, lung edema, histological abnormalities, and physiological dysfunction that characterize ALI. Using a cecal slurry (CS) model of polymicrobial abdominal sepsis and exposure to hyperoxia (95%), we systematically varied the timing and dose of the CS injection, fluids and antibiotics, and dose of hyperoxia. We found that CS alone had a high mortality rate that was improved with the addition of antibiotics and fluids. Despite this, we did not see evidence of ALI as measured by bronchoalveolar lavage (BAL) cell count, total protein, C-X-C motif chemokine ligand 1 (CXCL-1) or by lung wet:dry weight ratio. Addition of hyperoxia [95% fraction of inspired oxygen ([Formula: see text])] to CS immediately after CS injection increased BAL cell counts, CXCL-1, and lung wet:dry weight ratio but was associated with 40% mortality. Splitting the hyperoxia treatment into two 12-h exposures (0-12 h and 24-36 h) after CS injection increased survival to 75% and caused significant lung injury compared with CS alone as measured by increased BAL total cell count (92,500 vs. 240,000, P = 0.0004), BAL protein (71 vs. 103 µg/mL, P = 0.0030), and lung wet:dry weight ratio (4.5 vs. 5.5, P = 0.0005), and compared with sham as measured by increased BAL CXCL-1 (20 vs. 2,372 pg/mL, P < 0.0001) and histological lung injury score (1.9 vs. 4.2, P = 0.0077). In addition, our final model showed evidence of lung epithelial [increased BAL and plasma receptor for advanced glycation end products (RAGE)] and endothelial (increased Syndecan-1 and sulfated glycosaminoglycans) injury. In conclusion, we have developed a clinically relevant mouse model of sepsis-induced ALI using intraperitoneal injection of CS, antibiotics and fluids, and hyperoxia. This clinically relevant model can be used for future studies of sepsis-induced ALI.

A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus.

Multi-drug resistant Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), has become a worldwide, major health care problem. While initially restricted to clinical settings, drug resistant S. aureus is now one of the key causative agents of community-acquired infections. We have previously demonstrated that copper dependent inhibitors (CDIs), a class of antibiotics that are only active in the presence of copper ions, are effective bactericidal agents against MRSA. A second-generation CDI, APT-6K, exerted bactericidal activity at nanomolar concentrations. At sub-bactericidal concentrations, it effectively synergized with ampicillin to reverse drug resistance in multiple MRSA strains. APT-6K had a favorable therapeutic index when tested on eukaryotic cells (TI: > 30) and, unlike some previously reported CDIs, did not affect mitochondrial activity. These results further establish inhibitors that are activated by the binding of transition metal ions as a promising class of antibiotics, and for the first time, describe their ability to reverse existing drug resistance against clinically relevant antibiotics.

Lack of selective resistance of influenza A virus in presence of host-targeted antiviral, UV-4B.

Development of antiviral drug resistance is a continuous concern for viruses with high mutation rates such as influenza. The use of antiviral drugs targeting host proteins required for viral replication is less likely to result in the selection of resistant viruses than treating with direct-acting antivirals. The iminosugar UV-4B is a host-targeted glucomimetic that inhibits endoplasmic reticulum α-glucosidase I and II enzymes resulting in improper glycosylation and misfolding of viral glycoproteins. UV-4B has broad-spectrum antiviral activity against diverse viruses including dengue and influenza. To examine the ability of influenza virus to develop resistance against UV-4B, mouse-adapted influenza virus was passaged in mice in the presence or absence of UV-4B and virus isolated from lungs was used to infect the next cohort of mice, for five successive passages. Deep sequencing was performed to identify changes in the viral genome during passaging in the presence or absence of UV-4B. Relatively few minor variants were identified within each virus and the ratio of nonsynonymous to synonymous (dN/dS) substitutions of minor variants confirmed no apparent positive selection following sustained exposure to UV-4B. Three substitutions (one synonymous in PB2, one nonsynonymous in M and PA each) were specifically enriched (>3%) in UV-4B-treated groups at passage five. Recombinant viruses containing each individual or combinations of these nonsynonymous mutations remained sensitive to UV-4B treatment in mice. Overall, these data provide evidence that there is a high genetic barrier to the generation and selection of escape mutants following exposure to host-targeted iminosugar antivirals.

The phosphatase PPM1A controls monocyte-to-macrophage differentiation.

Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a critical regulatory process affecting host defense and inflammation. However, the regulatory signaling pathways that control the differentiation of monocytes into specific and distinct functional macrophage subsets are poorly understood. Herein, we demonstrate that monocyte-to-macrophage differentiation is controlled by the Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A). Genetic manipulation experiments demonstrated that overexpression of PPM1A attenuated the macrophage differentiation program, while knockdown of PPM1A expression accelerated the ability of monocytes to differentiate into macrophages. We identify imiquimod and Pam3CSK4 as two Toll-like receptor agonists that induce PPM1A expression, and show that increased expression of PPM1A at the onset of differentiation impairs cellular adherence, reduces expression of inflammatory (M1) macrophage-specific markers, and inhibits the production of inflammatory cytokines. Our findings reveal PPM1A as a negative threshold regulator of M1-type monocyte-to-macrophage differentiation, establishing it as a key phosphatase that orchestrates this program.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

Mycobacterium tuberculosis exploits the PPM1A signaling pathway to block host macrophage apoptosis.

The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly while protecting it from antibiotic treatment. We recently described that Mtb infection upregulated expression of the host phosphatase PPM1A, which impairs the antibacterial response of macrophages. Here we establish PPM1A as a checkpoint target used by Mtb to suppress macrophage apoptosis. Overproduction of PPM1A suppressed apoptosis of Mtb-infected macrophages by a mechanism that involves inactivation of the c-Jun N-terminal kinase (JNK). Targeted depletion of PPM1A by shRNA or inhibition of PPM1A activity by sanguinarine restored JNK activation, resulting in increased apoptosis of Mtb-infected macrophages. We also demonstrate that activation of JNK by subtoxic concentrations of anisomycin induced selective apoptotic killing of Mtb-infected human macrophages, which was completely blocked in the presence of a specific JNK inhibitor. Finally, selective killing of Mtb-infected macrophages and subsequent bacterial release enabled rifampicin to effectively kill Mtb at concentrations that were insufficient to act against intracellular Mtb, providing proof of principle for the efficacy of a "release and kill" strategy. Taken together, these findings suggest that drug-induced selective apoptosis of Mtb-infected macrophages is achievable.

Protein phosphatase, Mg2+/Mn2+-dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection.

Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated the ability of THP-1 cells to respond to relevant bacterial stimuli with a proper cytokine/chemokine secretion response, blocked their chemotactic response and impaired their ability to phagocytose bacteria. These data suggest that PPM1A, which had previously been shown to play a role in the antiviral response to Herpes Simplex virus infection, also governs the antibacterial response of macrophages to bacteria, or at least to Mtb infection. PPM1A thus seems to play a central role in the innate immune response of macrophages, implying that host directed therapies targeting PPM1A could be highly beneficial, in particular for HIV/Mtb co-infected patients.

Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity.

The continuous rise of multi-drug resistant pathogenic bacteria has become a significant challenge for the health care system. In particular, novel drugs to treat infections of methicillin-resistant Staphylococcus aureus strains (MRSA) are needed, but traditional drug discovery campaigns have largely failed to deliver clinically suitable antibiotics. More than simply new drugs, new drug discovery approaches are needed to combat bacterial resistance. The recently described phenomenon of copper-dependent inhibitors has galvanized research exploring the use of metal-coordinating molecules to harness copper's natural antibacterial properties for therapeutic purposes. Here, we describe the results of the first concerted screening effort to identify copper-dependent inhibitors of Staphylococcus aureus. A standard library of 10 000 compounds was assayed for anti-staphylococcal activity, with hits defined as those compounds with a strict copper-dependent inhibitory activity. A total of 53 copper-dependent hit molecules were uncovered, similar to the copper independent hit rate of a traditionally executed campaign conducted in parallel on the same library. Most prominent was a hit family with an extended thiourea core structure, termed the NNSN motif. This motif resulted in copper-dependent and copper-specific S. aureus inhibition, while simultaneously being well tolerated by eukaryotic cells. Importantly, we could demonstrate that copper binding by the NNSN motif is highly unusual and likely responsible for the promising biological qualities of these compounds. A subsequent chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, despite the family's presence in many chemical screening libraries. Thus, our copper-biased screen has proven able to discover inhibitors within previously screened libraries, offering a mechanism to reinvigorate exhausted molecular collections.

A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis.

In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that improve the drug development process will be essential to accelerate the development of next-generation antituberculosis drugs. The drug development process seems to be hampered by the inefficient transition of initially promising hits to candidate compounds that are effective in vivo. In this study, we introduce an inexpensive, rapid, and BSL-2 compatible infection model using macrophage-passaged Mycobacterium tuberculosis (Mtb) that forms densely packed Mtb/macrophage aggregate structures suitable for drug efficacy testing. Susceptibility to antituberculosis drugs determined with this Mtb/macrophage aggregate model differed from commonly used in vitro broth-grown single-cell Mtb cultures. Importantly, altered drug susceptibility correlated well with the reported ability of the respective drugs to generate high tissue and cerebrospinal fluid concentrations relative to their serum concentrations, which seems to be the best predictors of in vivo efficacy. Production of these Mtb/macrophage aggregates could be easily scaled up to support throughput efforts. Overall, its simplicity and scalability should make this Mtb/macrophage aggregate model a valuable addition to the currently available Mtb drug discovery tools.