Metabolic changes enhance necroptosis of type 2 diabetes mellitus mice infected with Mycobacterium tuberculosis.

Previously, we found that Mycobacterium tuberculosis (Mtb) infection in type 2 diabetes mellitus (T2DM) mice enhances inflammatory cytokine production which drives pathological immune responses and mortality. In the current study, using a T2DM Mtb infection mice model, we determined the mechanisms that make T2DM mice alveolar macrophages (AMs) more inflammatory upon Mtb infection. Among various cell death pathways, necroptosis is a major pathway involved in inflammatory cytokine production by T2DM mice AMs. Anti-TNFR1 antibody treatment of Mtb-infected AMs from T2DM mice significantly reduced expression of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) (necroptosis markers) and IL-6 production. Metabolic profile comparison of Mtb-infected AMs from T2DM mice and Mtb-infected AMs of nondiabetic control mice indicated that 2-ketohexanoic acid and deoxyadenosine monophosphate were significantly abundant, and acetylcholine and pyridoxine (Vitamin B6) were significantly less abundant in T2DM mice AMs infected with Mtb. 2-Ketohexanoic acid enhanced expression of TNFR1, RIPK3, MLKL and inflammatory cytokine production in the lungs of Mtb-infected nondiabetic mice. In contrast, pyridoxine inhibited RIPK3, MLKL and enhanced expression of Caspase 3 (apoptosis marker) in the lungs of Mtb-infected T2DM mice. Our findings demonstrate that metabolic changes in Mtb-infected T2DM mice enhance TNFR1-mediated necroptosis of AMs, which leads to excess inflammation and lung pathology.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Antibacterial peptide Reg4 ameliorates Pseudomonas aeruginosa-induced pulmonary inflammation and fibrosis.

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative facultative anaerobe that has become an important cause of severe infections in humans, particularly in patients with cystic fibrosis. The development of efficacious methods or mendicants against P. aeruginosa is still needed. We previously reported that regenerating islet-derived family member 4 (Reg4) has bactericidal activity against Salmonella Typhimurium, a Gram-negative flagellated bacterium. We herein explore whether Reg4 has bactericidal activity against P. aeruginosa. In the P. aeruginosa PAO1-chronic infection model, Reg4 significantly inhibits the colonization of PAO1 in the lung and subsequently ameliorates pulmonary inflammation and fibrosis. Reg4 recombinant protein suppresses the growth motility and biofilm formation capability of PAO1 in vitro. Mechanistically, Reg4 not only exerts bactericidal action via direct binding to the P. aeruginosa cell wall but also enhances the phagocytosis of alveolar macrophages in the host. Taken together, our study demonstrates that Reg4 may provide protection against P. aeruginosa-induced pulmonary inflammation and fibrosis via its antibacterial activity.IMPORTANCEChronic lung infection with Pseudomonas aeruginosa is a leading cause of morbidity and mortality in patients with cystic fibrosis. Due to the antibiotic resistance of Pseudomonas aeruginosa, antimicrobial peptides appear to be a potential alternative to combat its infection. In this study, we report an antimicrobial peptide, regenerating islet-derived 4 (Reg4), that showed killing activity against clinical strains of Pseudomonas aeruginosa PAO1 and ameliorated PAO1-induced pulmonary inflammation and fibrosis. Experimental data also showed Reg4 directly bound to the bacterial cell membrane and enhanced the phagocytosis of host alveolar macrophages. Our presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Comparative proteome analysis revealed the differences in response to both Mycobacterium tuberculosis and Mycobacterium bovis infection of bovine alveolar macrophages.

Tuberculosis (TB), attributed to the Mycobacterium tuberculosis complex, is one of the most serious zoonotic diseases worldwide. Nevertheless, the host mechanisms preferentially leveraged by Mycobacterium remain unclear. After infection, both Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (MB) bacteria exhibit intimate interactions with host alveolar macrophages; however, the specific mechanisms underlying these macrophage responses remain ambiguous. In our study, we performed a comparative proteomic analysis of bovine alveolar macrophages (BAMs) infected with MTB or MB to elucidate the differential responses of BAMs to each pathogen at the protein level. Our findings revealed heightened TB infection susceptibility of BAMs that had been previously infected with MTB or MB. Moreover, we observed that both types of mycobacteria triggered significant changes in BAM energy metabolism. A variety of proteins and signalling pathways associated with autophagy and inflammation-related progression were highly activated in BAMs following MB infection. Additionally, proteins linked to energy metabolism were highly expressed in BAMs following MTB infection. In summary, we propose that BAMs may resist MTB and MB infections via different mechanisms. Our findings provide critical insights into TB pathogenesis, unveiling potential biomarkers to facilitate more effective TB treatment strategies. Additionally, our data lend support to the hypothesis that MTB may be transmitted via cross-species infection.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

The Dual Benefit of Sulfasalazine on Pneumocystis Pneumonia-Related Immunopathogenesis and Antifungal Host Defense Does Not Require IL-4Rα-Dependent Macrophage Polarization.

Pneumocystis is a respiratory fungal pathogen that is among the most frequent causes of life-threatening pneumonia (PcP) in immunocompromised hosts. Alveolar macrophages play an important role in host defense against Pneumocystis, and several studies have suggested that M2 polarized macrophages have anti-Pneumocystis effector activity. Our prior work found that the immunomodulatory drug sulfasalazine (SSZ) provides a dual benefit during PcP-related immune reconstitution inflammatory syndrome (IRIS) by concurrently suppressing immunopathogenesis while also accelerating macrophage-mediated fungal clearance. The benefits of SSZ were associated with heightened Th2 cytokine production and M2 macrophage polarization. Therefore, to determine whether SSZ improves the outcome of PcP through a mechanism that requires Th2-dependent M2 polarization, RAG2-/- mice lacking interleukin 4 receptor alpha chain (IL-4Rα) on macrophage lineage cells were generated. As expected, SSZ treatment dramatically reduced the severity of PcP-related immunopathogenesis and accelerated fungal clearance in immune-reconstituted RAG2-/- mice. Similarly, SSZ treatment was also highly effective in immune-reconstituted RAG2/IL-4Rα-/- and RAG2/gamma interferon receptor (IFN-γR)-/- mice, demonstrating that neither IL-4Rα-dependent M2 nor IFN-γR-dependent M1 macrophage polarization programs were required for the beneficial effects of SSZ. Despite the fact that macrophages from RAG2/IL-4Rα-/- mice could not respond to the Th2 cytokines IL-4 and IL-13, M2-biased alveolar macrophages were identified in the lungs following SSZ treatment. These data demonstrate that not only does SSZ enhance phagocytosis and fungal clearance in the absence of macrophage IL-4Rα signaling, but also that SSZ promotes M2 macrophage polarization in an IL-4Rα-independent manner. These findings could have implications for the treatment of PcP and other diseases in which M2 polarization is beneficial.

Mycobacterium tuberculosis-macrophage interaction: Molecular updates.

Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB), remains a pathogen of great interest on a global scale. This airborne pathogen affects the lungs, where it interacts with macrophages. Acidic pH, oxidative and nitrosative stressors, and food restrictions make the macrophage's internal milieu unfriendly to foreign bodies. Mtb subverts the host immune system and causes infection due to its genetic arsenal and secreted effector proteins. In vivo and in vitro research have examined Mtb-host macrophage interaction. This interaction is a crucial stage in Mtb infection because lung macrophages are the first immune cells Mtb encounters in the host. This review summarizes Mtb effectors that interact with macrophages. It also examines how macrophages control and eliminate Mtb and how Mtb manipulates macrophage defense mechanisms for its own survival. Understanding these mechanisms is crucial for TB prevention, diagnosis, and treatment.

Apoptosis Inhibitor of Macrophages Contributes to the Chronicity of Mycobacterium avium Infection by Promoting Foamy Macrophage Formation.

In Mycobacterium avium infections, macrophages play a critical role in the host defense response. Apoptosis inhibitor of macrophage (AIM), also known as CD5L, may represent a novel supportive therapy against various diseases, including metabolic syndrome and infectious diseases. The mechanisms of AIM include modulating lipid metabolism in macrophages and other host cells. We investigated the role of AIM in M. avium infections in vitro and in vivo. In a mouse model of M. avium pneumonia, foamy macrophages were induced 6 wk after infection. The bacteria localized in these macrophages. Flow cytometric analysis also confirmed that the percentage of CD11chighMHCclassIIhigh interstitial and alveolar macrophages, a cell surface marker defined as foamy macrophages, increased significantly after infection. AIM in alveolar lavage fluid and serum gradually increased after infection. Administration of recombinant AIM significantly increased the number of bacteria in the lungs of mice, accompanied by the induction of inflammatory cytokine and iNOS expression. In mouse bone marrow-derived macrophages, the mRNA expression of AIM after M. avium infection and the amount of AIM in the supernatant increased prior to the increase in intracellular bacteria. Infected cells treated with anti-AIM Abs had fewer bacteria and a higher percentage of apoptosis-positive cells than infected cells treated with isotype control Abs. Finally, AIM in the sera of patients with M. avium-pulmonary disease was measured and was significantly higher than in healthy volunteers. This suggests that AIM production is enhanced in M. avium-infected macrophages, increasing macrophage resistance to apoptosis and providing a possible site for bacterial growth.

Resistin secreted by porcine alveolar macrophages leads to endothelial cell dysfunction during Haemophilus parasuis infection.

Haemophilus parasuis (H. parasuis) causes exudative inflammation, implying endothelial dysfunction during pathogen infection. However, so far, the molecular mechanism of endothelial dysfunction caused by H. parasuis has not been clarified. By using the transwell-based cell co-culture system, we demonstrate that knocking out resistin in porcine alveolar macrophages (PAMs) dramatically attenuated endothelial monolayer damage caused by H. parasuis. The resistin secreted by PAMs inhibited the expression of the tight junction proteins claudin-5 and occludin rather than the adherens junction protein VE-cadherin in co-cultured porcine aortic endothelial cells (PAECs). Furthermore, we demonstrate that resistin regulated claudin-5 and occludin expression and monolayer PAEC permeability in an LKB1/AMPK/mTOR pathway-dependent manner. Additionally, we reveal that the outer membrane lipoprotein gene lppA in H. parasuis induced resistin expression in PAMs, as deleting lppA reduced resistin expression in H. parasuis-infected PAMs, causing a significant change in LKB1/AMPK/mTOR pathway activity in co-cultured PAECs, thereby restoring tight junction protein levels and endothelial monolayer permeability. Thus, we postulate that the H. parasuis lppA gene enhances resistin production in PAMs, disrupting tight junctions in PAECs and causing endothelial barrier dysfunction. These findings elucidate the pathogenic mechanism of exudative inflammation caused by H. parasuis for the first time and provide a more profound angle of acute exudative inflammation caused by bacteria.

Lipidomics revealed the global lipid responses of primary bovine alveolar macrophages to infections of Mycobacterium tuberculosis and Mycobacterium bovis.

Mycobacterium tuberculosis (M. tuberculosis (MTB) and M. bovis (MB) of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of the notorious infectious disease tuberculosis (TB) in a range of mammals, including bovine and human. The lipid composition of MTB/MB performed imperative function as invading host macrophage. However, the detailed variations in lipid compositions of MTB and MB were unknown, while the responses relevant to lipid metabolisms in MTB/MB-infected host were also unclear. In the present study, a dual-Lipidomics were used to elucidate the differences in lipid composition of MTB and MB and responses in lipid metabolisms of primary bovine alveolar macrophages infected by MTB/MB. The Lipidomics showed significant differences in lipid composition, especially differences in levels of Glycerophospholipids, Sterol Lipids, Fatty Acyls and Polyketides between these two mycobacterium species. Meanwhile, both MTB and MB could invoke various responses of lipid metabolisms in host macrophages. An infection of MTB mainly induced the increases of Polyketides and Glycerophospholipids in macrophages, whereas an MB infection induced the increases of Glycerophospholipids and Sterol. Furthermore, TAG 13:0-18:5-18:5 of MTB and PC (16:1(9E)/0:0), PI(20:2(11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), 4, 6-Decadiyn-1-ol isovalerate and LacCer (d18:1/24:1(15Z)) of MB were identified to cause variations in lipid metabolisms of macrophages, respectively. From these data, we proposed that the differential compositions of lipid compositions in MTB and MB could successfully colonize in macrophage by different mechanisms. MTB could promote the formation of foam cells of macrophage for its colonization and development, while MB mainly suppresses the macrophage autophagy to escape the immune responses of host.

Anthocyanin Extract from Purple Sweet Potato Exacerbate Mitophagy to Ameliorate Pyroptosis in Klebsiella pneumoniae Infection.

Given the rise of morbidity and mortality caused by Klebsiella pneumoniae (KP), the increasing number of strains resistant to antibiotics, and the emergence of hypervirulent Klebsiella pneumonia, treatment of KP infection becomes difficult; thus, novel drugs are necessary for treatment. Anthocyanins, or natural flavonoids, have an extensive effect against bacterial infection. However, few studies on anti-KP are identified. Here, we evaluated the therapeutic effect of purple sweet potato anthocyanins (PSPAs) on KP, containing 98.7% delphinidin 3-sambubioside. Results showed that KP-infected mice after PSPAs treatment manifested decreased mortality, weakened lung injury, dampened inflammatory responses, and reduced bacterial systemic dissemination in vivo. In Vitro, PSPAs significantly suppressed pyroptosis and restricted NLRP3 inflammasome activation in alveolar macrophages infected with KP. As for the mechanism, PSPAs promote mitophagy by recruiting Parkin to the mitochondria. PSPAs-conferred mitophagy increased mitochondrial membrane potential and decreased mitochondrial reactive oxygen species and mitochondrial DNA, resulting in impaired NLRP3 inflammasome activation. In addition, the promotion of mitophagy by PSPAs required the Nrf2 signaling pathway. Collectively, these findings suggest that PSPAs are a potential option for the treatment of KP infection.

Type I Interferons Are Involved in the Intracellular Growth Control of Mycobacterium abscessus by Mediating NOD2-Induced Production of Nitric Oxide in Macrophages.

Mycobacterium abscessus (MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-ß treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/ß receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-ß treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-ß before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-ß did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-ß gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-ß treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.

Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production.

Tuberculosis is a deadly, contagious respiratory disease that is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb is adept at manipulating and evading host immunity by hijacking alveolar macrophages, the first line of defense against inhaled pathogens, by regulating the mode and timing of host cell death. It is established that Mtb infection actively blocks apoptosis and instead induces necrotic-like modes of cell death to promote disease progression. This survival strategy shields the bacteria from destruction by the immune system and antibiotics while allowing for the spread of bacteria at opportunistic times. As such, it is critical to understand how Mtb interacts with host macrophages to manipulate the mode of cell death. Herein, we demonstrate that Mtb infection triggers a time-dependent reduction in the expression of focal adhesion kinase (FAK) in human macrophages. Using pharmacological perturbations, we show that inhibition of FAK (FAKi) triggers an increase in a necrotic form of cell death during Mtb infection. In contrast, genetic overexpression of FAK (FAK+) completely blocked macrophage cell death during Mtb infection. Using specific inhibitors of necrotic cell death, we show that FAK-mediated cell death during Mtb infection occurs in a RIPK1-depedent, and to a lesser extent, RIPK3-MLKL-dependent mechanism. Consistent with these findings, FAKi results in uncontrolled replication of Mtb, whereas FAK+ reduces the intracellular survival of Mtb in macrophages. In addition, we demonstrate that enhanced control of intracellular Mtb replication by FAK+ macrophages is a result of increased production of antibacterial reactive oxygen species (ROS) as inhibitors of ROS production restored Mtb burden in FAK+ macrophages to same levels as in wild-type cells. Collectively, our data establishes FAK as an important host protective response during Mtb infection to block necrotic cell death and induce ROS production, which are required to restrict the survival of Mtb.

Vaccination induces rapid protection against bacterial pneumonia via training alveolar macrophage in mice.

Vaccination strategies for rapid protection against multidrug-resistant bacterial infection are very important, especially for hospitalized patients who have high risk of exposure to these bacteria. However, few such vaccination strategies exist due to a shortage of knowledge supporting their rapid effect. Here, we demonstrated that a single intranasal immunization of inactivated whole cell of Acinetobacter baumannii elicits rapid protection against broad A. baumannii-infected pneumonia via training of innate immune response in Rag1-/- mice. Immunization-trained alveolar macrophages (AMs) showed enhanced TNF-α production upon restimulation. Adoptive transfer of immunization-trained AMs into naive mice mediated rapid protection against infection. Elevated TLR4 expression on vaccination-trained AMs contributed to rapid protection. Moreover, immunization-induced rapid protection was also seen in Pseudomonas aeruginosa and Klebsiella pneumoniae pneumonia models, but not in Staphylococcus aureus and Streptococcus pneumoniae model. Our data reveal that a single intranasal immunization induces rapid and efficient protection against certain Gram-negative bacterial pneumonia via training AMs response, which highlights the importance and the possibility of harnessing trained immunity of AMs to design rapid-effecting vaccine.

Contribution of dipeptidyl peptidase 4 to non-typeable Haemophilus influenzae-induced lung inflammation in COPD.

Dipeptidyl peptidase 4 (DPP4) expression is increased in the lungs of chronic obstructive pulmonary disease (COPD). DPP4 is known to be associated with inflammation in various organs, including LPS-induced acute lung inflammation. Since non-typeable Haemophilus influenzae (NTHi) causes acute exacerbations in COPD patients, we examined the contribution of DPP4 in NTHi-induced lung inflammation in COPD. Pulmonary macrophages isolated from COPD patients showed higher expression of DPP4 than the macrophages isolated from normal subjects. In response to NTHi infection, COPD, but not normal macrophages show a further increase in the expression of DPP4. COPD macrophages also showed higher expression of IL-1ß, and CCL3 responses to NTHi than normal, and treatment with DPP4 inhibitor, diprotin A attenuated this response. To examine the contribution of DPP4 in NTHi-induced lung inflammation, COPD mice were infected with NTHi, treated with diprotin A or PBS intraperitoneally, and examined for DPP4 expression, lung inflammation, and cytokine expression. Mice with COPD phenotype showed increased expression of DPP4, which increased further following NTHi infection. DPP4 expression was primarily observed in the infiltrated inflammatory cells. NTHi-infected COPD mice also showed sustained neutrophilic lung inflammation and expression of CCL3, and this was inhibited by DPP4 inhibitor. These observations indicate that enhanced expression of DPP4 in pulmonary macrophages may contribute to sustained lung inflammation in COPD following NTHi infection. Therefore, inhibition of DPP4 may reduce the severity of NTHi-induced lung inflammation in COPD.

Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung.

In this study, we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression, and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis-infected alveolar (AMs) and interstitial macrophages (IMs) in vivo. We identify clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. This conceptual approach is readily transferable to other infectious disease agents with the potential for an increased understanding of the roles that different host cell populations play during the course of an infection.

Macrophage Infection Models for Mycobacterium tuberculosis.

Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages, HoxB8 conditionally immortalized myeloid cells, Max Planck Institute alveolar macrophage-like cells, and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.