Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3.

BACKGROUND: Helicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis. METHODS: The expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry. RESULTS: After H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased. CONCLUSIONS: In summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis.Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence.Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response.Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages.Results. We demonstrated that the deletion of the gene codY resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner.Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.

Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles.

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Biofilms and core pathogens shape the tumor microenvironment and immune phenotype in colorectal cancer.

Extensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4+ T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

Roles of airway and intestinal epithelia in responding to pathogens and maintaining tissue homeostasis.

Epithelial cells form a resilient barrier and orchestrate defensive and reparative mechanisms to maintain tissue stability. This review focuses on gut and airway epithelia, which are positioned where the body interfaces with the outside world. We review the many signaling pathways and mechanisms by which epithelial cells at the interface respond to invading pathogens to mount an innate immune response and initiate adaptive immunity and communicate with other cells, including resident microbiota, to heal damaged tissue and maintain homeostasis. We compare and contrast how airway and gut epithelial cells detect pathogens, release antimicrobial effectors, collaborate with macrophages, Tregs and epithelial stem cells to mount an immune response and orchestrate tissue repair. We also describe advanced research models for studying epithelial communication and behaviors during inflammation, tissue injury and disease.

Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by regulating cAMP level.

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.

Integrating machine learning algorithms and single-cell analysis to identify gut microbiota-related macrophage biomarkers in atherosclerotic plaques.

Objective: The relationship between macrophages and the gut microbiota in patients with atherosclerosis remains poorly defined, and effective biological markers are lacking. This study aims to elucidate the interplay between gut microbial communities and macrophages, and to identify biomarkers associated with the destabilization of atherosclerotic plaques. The goal is to enhance our understanding of the underlying molecular pathways and to pave new avenues for diagnostic approaches and therapeutic strategies in the disease. Methods: This study employed Weighted Gene Co-expression Network Analysis (WGCNA) and differential expression analysis on atherosclerosis datasets to identify macrophage-associated genes and quantify the correlation between these genes and gut microbiota gene sets. The Random Forest algorithm was utilized to pinpoint PLEK, IRF8, BTK, CCR1, and CD68 as gut microbiota-related macrophage genes, and a nomogram was constructed. Based on the top five genes, a Non-negative Matrix Factorization (NMF) algorithm was applied to construct gut microbiota-related macrophage clusters and analyze their potential biological alterations. Subsequent single-cell analyses were conducted to observe the expression patterns of the top five genes and the interactions between immune cells. Finally, the expression profiles of key molecules were validated using clinical samples from atherosclerosis patients. Results: Utilizing the Random Forest algorithm, we ultimately identified PLEK, IRF8, CD68, CCR1, and BTK as gut microbiota-associated macrophage genes that are upregulated in atherosclerotic plaques. A nomogram based on the expression of these five genes was constructed for use as an auxiliary tool in clinical diagnosis. Single-cell analysis confirmed the specific expression of gut microbiota-associated macrophage genes in macrophages. Clinical samples substantiated the high expression of PLEK in unstable atherosclerotic plaques. Conclusion: Gut microbiota-associated macrophage genes (PLEK, IRF8, CD68, CCR1, and BTK) may be implicated in the pathogenesis of atherosclerotic plaques and could serve as diagnostic markers to aid patients with atherosclerosis.

Targeting Mycobacterium tuberculosis pH-driven adaptation.

Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.

HDAC6 inhibitor promotes reactive oxygen species-meditated clearance of Staphylococcus aureus in macrophage.

Staphylococcus aureus (S. aureus) pneumonia has become an increasingly important public health problem. Recent evidence suggests that epigenetic modifications are critical in the host immune defence against pathogen infection. In this study, we found that S. aureus infection induces the expression of histone deacetylase 6 (HDAC6) in a dose-dependent manner. Furthermore, by using a S. aureus pneumonia mouse model, we showed that the HDAC6 inhibitor, tubastatin A, demonstrates a protective effect in S. aureus pneumonia, decreasing the mortality and destruction of lung architecture, reducing the bacterial burden in the lungs and inhibiting inflammatory responses. Mechanistic studies in primary bone marrow-derived macrophages demonstrated that the HDAC6 inhibitors, tubastatin A and tubacin, reduced the intracellular bacterial load by promoting bacterial clearance rather than regulating phagocytosis. Finally, N-acetyl-L- cysteine, a widely used reactive oxygen species (ROS) scavenger, antagonized ROS production and significantly inhibited tubastatin A-induced S. aureus clearance. These findings demonstrate that HDAC6 inhibitors promote the bactericidal activity of macrophages by inducing ROS, an important host factor for S. aureus clearance and production. Our study identified HDAC6 as a suitable epigenetic modification target for preventing S. aureus infection, and tubastatin A as a useful compound in treating S. aureus pneumonia.

Cystatin C: immunoregulation role in macrophages infected with Porphyromonas gingivalis.

Background: Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. Porphyromonas gingivalis is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of P. gingivalis. This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with P. gingivalis. Methods: Monocyte-derived macrophages generated from peripheral blood were infected with P. gingivalis (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of P. gingivalis and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined. Results: Our results showed that cystatin C inhibits the extracellular growth of P. gingivalis. In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis. Conclusions: Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with P. gingivalis. These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis.

Lycopene promoted M2 macrophage polarization via inhibition of NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to Escherichia coli infection in J744A.1 cells.

Escherichia coli (E. coli) can induce severe clinical bovine mastitis, which is to blame for large losses experienced by dairy farms. Macrophage polarization into various states is in response to pathogen infections. Lycopene, a naturally occurring hydrocarbon carotenoid, relieved inflammation by controlling M1/M2 status of macrophages. Thus, we wanted to explore the effect of lycopene on polarization states of macrophages in E. coli-induced mastitis. Macrophages were cultivated with lycopene for 24, before E. coli inoculation for 6 h. Lycopene (0.5 µmol/L) significantly enhanced cell viabilities and significantly reduced lactic dehydrogenase (LDH) levels in macrophages, whereas 2 and 3 µmol/L lycopene significantly enhanced LDH activities. Lycopene treatment significantly reduced the increase in LDH release, iNOS, CD86, TNF-α, IL-1ß and phosphatase and tensin homolog (PTEN) expressions in E. coli group. 0.5 µmol/L lycopene significantly increased E. coli-induced downregulation of CD206, arginase I (ARG1), indoleamine 2,3-dioxygenase (IDO), chitinase 3-like 3 (YM1), PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, jumonji domain-containing protein-3 (JMJD3) and interferon regulatory factor 4 (IRF4) levels. Moreover, Ginkgolic acid C17:1 (a specific PTEN inhibitor), 740YPDGFR (a specific PI3K activator), SC79 (a specific AKT activator) or CHPG sodium salt (a specific NF-κB activator) significantly decreased CD206, AGR1, IDO and YM1 expressions in lycopene and E. coli-treated macrophages. Therefore, lycopene increased M2 macrophages via inhibiting NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to E. coli infection in macrophages. These results contribute to revealing the pathogenesis of E. coli-caused bovine mastitis, providing the new angle of the prevention and management of mastitis.

Transcriptome analysis of long non-coding RNAs in Mycobacterium avium complex-infected macrophages.

Mycobacterium avium complex (MAC) is a non-tuberculous mycobacterium widely distributed in the environment. Even though MAC infection is increasing in older women and immunocompromised patients, to our knowledge there has been no comprehensive analysis of the MAC-infected host-cell transcriptome-and particularly of long non-coding RNAs (lncRNAs). By using in vitro-cultured primary mouse bone-marrow-derived macrophages (BMDMs) and Cap analysis of gene expression, we analyzed the transcriptional and kinetic landscape of macrophage genes, with a focus on lncRNAs, during MAC infection. MAC infection of macrophages induced the expression of immune/inflammatory response genes and other genes similar to those involved in M1 macrophage activation, consistent with previous reports, although Nos2 (M1 activation) and Arg1 (M2 activation) had distinct expression profiles. We identified 31 upregulated and 30 downregulated lncRNA promoters corresponding respectively to 18 and 26 lncRNAs. Upregulated lncRNAs were clustered into two groups-early and late upregulated-predicted to be associated with immune activation and the immune response to infection, respectively. Furthermore, an Ingenuity Pathway Analysis revealed canonical pathways and upstream transcription regulators associated with differentially expressed lncRNAs. Several differentially expressed lncRNAs reported elsewhere underwent expressional changes upon M1 or M2 preactivation and subsequent MAC infection. Finally, we showed that expressional change of lncRNAs in MAC-infected BMDMs was mediated by toll-like receptor 2, although there may be other mechanisms that sense MAC infection. We identified differentially expressed lncRNAs in MAC-infected BMDMs, revealing diverse features that imply the distinct roles of these lncRNAs in MAC infection and macrophage polarization.

M2 macrophages participate in ILC2 activation induced by Helicobacter pylori infection.

Helicobacter pylori (H. pylori) causes a diversity of gastric diseases. The host immune response evoked by H. pylori infection is complicated and can influence the development and progression of diseases. We have reported that the Group 2 innate lymphocytes (ILC2) were promoted and took part in building type-2 immunity in H. pylori infection-related gastric diseases. Therefore, in the present study, we aim to clarify how H. pylori infection induces the activation of ILC2. It was found that macrophages were necessary for activating ILC2 in H. pylori infection. Mechanistically, H. pylori infection up-regulated the expression of indoleamine 2,3-dioxygenase (IDO) in macrophages to induce M2 polarization, and the latter secreted the alarmin cytokine Thymic Stromal Lymphopoietin (TSLP) to arouse ILC2.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

MicroRNA-144-3p Inhibits Host Lipid Catabolism and Autophagy by Targeting PPARα and ABCA1 During Mycobacterium Tuberculosis Infection.

MicroRNA-mediated metabolic reprogramming recently has been identified as an important strategy for Mycobacterium tuberculosis (Mtb) to evade host immune responses. However, it is unknown what role microRNA-144-3p (miR-144-3p) plays in cellular metabolism during Mtb infection. Here, we report the meaning of miR-144-3p-mediated lipid accumulation for Mtb-macrophage interplay. Mtb infection was shown to upregulate the expression of miR-144-3p in macrophages. By targeting peroxisome proliferator-activated receptor α (PPARα) and ATP-binding cassette transporter A1 (ABCA1), miR-144-3p overexpression promoted lipid accumulation and bacterial survival in Mtb-infected macrophages, while miR-144-3p inhibition had the opposite effect. Furthermore, reprogramming of host lipid metabolism by miR-144-3p suppressed autophagy in response to Mtb infection. Our findings uncover that miR-144-3p regulates host metabolism and immune responses to Mtb by targeting PPARα and ABCA1, suggesting a potential host-directed tuberculosis therapy by targeting the interface of miRNA and lipid metabolism.

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania.

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1ß, and IL-1ß compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.

The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in phagocytes.

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.

Insights into the effect of guanylate-binding protein 1 on the survival of Brucella intracellularly.

Brucellosis is a zoonotic disease that affects wild and domestic animals. It is caused by members of the bacterial genus Brucella. Guanylate-binding protein 1 (GBP1) is associated with microbial infections. However, the role of GBP1 during Brucella infection remains unclear. This investigation aimed to identify the association of GBP1 with brucellosis. Results showed that Brucella infection induced GBP1 upregulation in RAW 264.7 murine macrophages. Small interfering GBP1 targeting RNAs were utilized to explore how GBP1 regulates the survival of Brucella intracellularly. Results revealed that GBP1 knockdown promoted Brucella's survival ability, activated Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammatory corpuscles, and induced pro-inflammatory cytokines IFN-γ and IL-1ß. Furthermore, Brucella stimulated the expression of GBP1 in bone marrow-derived macrophages (BMDMs) and mice. During the inhibition of GBP1 in BMDMs, the intracellular growth of Brucella increased. In comparison, GBP1 downregulation enhanced the accumulation of Brucella-induced reactive oxygen species (ROS) in macrophages. Overall, the data indicate a significant role of GBP1 in regulating brucellosis and suggest the function underlying its suppressive effect on the survival and growth of Brucella intracellularly.