Macrophage Transcriptional Response Research Articles

The early bird catches the IL-2 in C-type lectin receptor-dependent activation of dendritic cells.

The transcriptional response of macrophages and dendritic cells to stimulation of pattern recognition receptors can be unexpectedly distinct. In this issue of Science Signaling, Watanabe etal. demonstrate that IL-2 is differentially induced by the closely related C-type lectin receptors Dectin-2 and Mincle and reveal early signaling through the adaptor protein FcRγ as a critical mechanism.

Exon 2-mediated deletion of Trem2 does not worsen metabolic function in diet-induced obese mice.

Triggering receptor expressed on myeloid cells 2 (Trem2) is highly expressed on myeloid cells and is involved in cellular lipid homeostasis and inflammatory processes. Trem2 deletion in mice (Trem2-/- ) evokes adipose tissue dysfunction, but its role in worsening obesity-induced metabolic dysfunction has not been resolved. Here we aimed to determine the causal role of Trem2 in regulating glucose homeostasis and insulin sensitivity in mice. Nine-week-old male and female littermate wild-type (WT) and Trem2-/- mice were fed a low- or high-fat diet for 18weeks and phenotyped for metabolic function. Diet-induced weight gain was similar between genotypes, irrespective of sex. Consistent with previous reports, we find that loss of Trem2 causes massive adipocyte hypertrophy and an attenuation in the lipid-associated macrophage transcriptional response to obesity. In contrast to published data, we find that loss of Trem2 does not worsen metabolic function in obese mice. No differences in intraperitoneal glucose tolerance (ipGTT), oral GTT or mixed meal substrate control, including postprandial glucose, non-esterified fatty acids, insulin or triglycerides, were found between WT and Trem2-/- animals. Similarly, no phenotypic differences existed when animals were challenged with stressors on metabolic demand (i.e. acute exercise or environmental temperature modulation). Collectively, we report a disassociation between adipose tissue remodelling caused by loss of Trem2 and whole-body metabolic homeostasis in obese mice. The complementary nature of experiments conducted gives credence to the conclusion that loss of Trem2 is unlikely to worsen glucose homeostasis in mice.

Mycobacterium tuberculosis canonical virulence factors interfere with a late component of the TLR2 response.

For many intracellular pathogens, the phagosome is the site of events and interactions that shape infection outcome. Phagosomal membrane damage, in particular, is proposed to benefit invading pathogens. To define the innate immune consequences of this damage, we profiled macrophage transcriptional responses to wild-type Mycobacterium tuberculosis (Mtb) and mutants that fail to damage the phagosomal membrane. We identified a set of genes with enhanced expression in response to the mutants. These genes represented a late component of the TLR2-dependent transcriptional response to Mtb, distinct from an earlier component that included Tnf. Expression of the later component was inherent to TLR2 activation, dependent upon endosomal uptake, and enhanced by phagosome acidification. Canonical Mtb virulence factors that contribute to phagosomal membrane damage blunted phagosome acidification and undermined the endosome-specific response. Profiling cell survival and bacterial growth in macrophages demonstrated that the attenuation of these mutants is partially dependent upon TLR2. Further, TLR2 contributed to the attenuated phenotype of one of these mutants in a murine model of infection. These results demonstrate two distinct components of the TLR2 response and identify a component dependent upon endosomal uptake as a point where pathogenic bacteria interfere with the generation of effective inflammation. This interference promotes tuberculosis (TB) pathogenesis in both macrophage and murine infection models.

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Transcriptional Analyses Identify Genes That Modulate Bovine Macrophage Response to Toxoplasma Infection and Immune Stimulation.

The obligate intracellular parasite, Toxoplasma gondii, is highly prevalent among livestock species. Although cattle are generally resistant to Toxoplasma strains circulating in Europe and North America, the underlying mechanisms are largely unknown. Here, we report that bovine bone marrow-derived macrophage (BMDM) pre-stimulated with interferon gamma (IFNγ) restricts intracellular Toxoplasma growth independently of nitric oxide. While Toxoplasma promoted the expression of genes associated with alternative macrophage activation and lipid metabolism, IFNγ abrogated parasite-induced transcriptional responses and promoted the expression of genes linked to the classical macrophage activation phenotype. Additionally, several chemokines, including CCL22, that are linked to parasite-induced activation of the Wnt/β-catenin signaling were highly expressed in Toxoplasma-exposed naïve BMDMs. A chemical Wnt/β-catenin signaling pathway antagonist (IWR-1-endo) significantly reduced intracellular parasite burden in naïve BMDMs, suggesting that Toxoplasma activates this pathway to evade bovine macrophage anti-parasitic responses. Congruently, intracellular burden of a mutant Toxoplasma strain (RHΔASP5) that does not secrete dense granule proteins into the host cell, which is an essential requirement for parasite-induced activation of the Wnt/β-catenin pathway, was significantly reduced in naïve BMDMs. However, both the Wnt/β-catenin antagonist and RHASPΔ5 did not abolish parasite burden differences in naïve and IFNγ-stimulated BMDMs. Finally, we observed that parasites infecting IFNγ-stimulated BMDMs largely express genes associated with the slow dividing bradyzoite stage. Overall, this study provides novel insights into bovine macrophage transcriptional response to Toxoplasma. It establishes a foundation for a mechanistic analysis IFNγ-induced bovine anti-Toxoplasma responses and the counteracting Toxoplasma survival strategies.

Cooperative and distinct functions of MK2 and MK3 in the regulation of the macrophage transcriptional response to lipopolysaccharide

The p38MAPK downstream targets MAPKAP kinases (MK) 2 and 3 are critical for the regulation of the macrophage response to LPS. The extents to which these two kinases act cooperatively and distinctly in regulating LPS-induced inflammatory cytokine expression are still unclear. To address this uncertainty, whole transcriptome analyses were performed using bone marrow-derived macrophages (BMDM) generated from MK2−/− or MK2/3−/− animals and their wild-type littermates. The results suggest that in BMDM, MK2 and MK3 not only cooperatively regulate the transcript expression of signaling intermediates, including IL-10, IL-19, CXCL2 and the IL-4 receptor (IL-4R)α subunit, they also exert distinct regulatory effects on the expression of specific transcripts. Based on the differential regulation of gene expression by MK2 and MK3, at least six regulatory patterns were identified. Importantly, we confirmed our previous finding, which showed that in the absence of MK2, MK3 negatively regulates IFN-β. Moreover, this genome-wide analysis identified the regulation of Cr1A, NOD1 and Serpina3f as similar to that of IFN-β. In the absence of MK2, MK3 also delayed the nuclear translocation of NFκB by delaying the ubiquitination and subsequent degradation of IκBβ, reflecting the substantial plasticity of the response of BMDM to LPS.

Influenza "Trains" the Host for Enhanced Susceptibility to Secondary Bacterial Infection.

We previously reported that the Toll-like receptor 4 (TLR4) antagonist Eritoran blocks acute lung injury (ALI) therapeutically in mouse and cotton rat models of influenza. However, secondary (2°) bacterial infection following influenza virus infection is associated with excess morbidity and mortality. Wild-type (WT) mice infected with mouse-adapted influenza A/Puerto Rico/8/34 virus (PR8) and, 7 days later, with Streptococcus pneumoniae serotype 3 (Sp3) exhibited significantly enhanced lung pathology and lethality that was reversed by Eritoran therapy after PR8 infection but before Sp3 infection. Cotton rats infected with nonadapted pH1N1 influenza virus and then superinfected with methicillin-resistant Staphylococcus aureus also exhibited increased lung pathology and serum high-mobility-group box 1 (HMGB1) levels, both of which were blunted by Eritoran therapy. In mice, PR8 infection suppressed Sp3-induced CXCL1 and CXCL2 mRNA, reducing neutrophil infiltration and increasing the bacterial burden, all of which were reversed by Eritoran treatment. While beta interferon (IFN-β)-deficient (IFN-β-/-) mice are highly susceptible to PR8, they exhibited delayed death upon Sp3 superinfection, indicating that while IFN-β was protective against influenza, it negatively impacted the host response to Sp3 IFN-β-treated WT macrophages selectively suppressed Sp3-induced CXCL1/CXCL2 transcriptionally, as evidenced by reduced recruitment of RNA polymerase II to the CXCL1 promoter. Thus, influenza establishes a "trained" state of immunosuppression toward 2° bacterial infection, in part through the potent induction of IFN-β and its downstream transcriptional regulation of chemokines, an effect reversed by Eritoran.IMPORTANCE Enhanced susceptibility to 2° bacterial infections following infection with influenza virus is a global health concern that accounts for many hospitalizations and deaths, particularly during pandemics. The complexity of the impaired host immune response during 2° bacterial infection has been widely studied. Both type I IFN and neutrophil dysfunction through decreased chemokine production have been implicated as mechanisms underlying enhanced susceptibility to 2° bacterial infections. Our findings support the conclusion that selective suppression of CXCL1/CXCL2 represents an IFN-β-mediated "training" of the macrophage transcriptional response to TLR2 agonists and that blocking of TLR4 therapeutically with Eritoran after influenza virus infection reverses this suppression by blunting influenza-induced IFN-β.

Conservation of Intracellular Pathogenic Strategy among Distantly Related Cryptococcal Species.

The genus Cryptococcus includes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to be Cryptococcus neoformans and Cryptococcus gattii We compared the interaction of murine macrophages with three C. gattii species complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and one C. neoformans species complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis. The most striking result was that the intracellular pathogenic strategies of C. neoformans and C. gattii species complex strains were qualitatively similar, despite the species having separated an estimated 100 million years ago. Macrophages exhibited a leaky phagolysosomal membrane phenotype and nonlytic exocytosis when infected with either C. gattii or C. neoformans Conservation of the intracellular strategy among species that separated long ago suggests that it is ancient and possibly maintained by similar selection pressures through eons.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response

A family of 11 cell surface-associated aspartyl proteases (CgYps1–11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata. However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1–11Δ mutant. Consistently, the mutant contained lower β-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1–11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1β in THP-1 macrophages. Further, Cgyps1–11Δ–induced IL-1β production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1–11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1β production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1–11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1β production in macrophages.

Enterococcus faecalis Promotes Innate Immune Suppression and Polymicrobial Catheter-Associated Urinary Tract Infection.

Enterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-κB signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coliE. faecalis and E. coli coinfection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to that with E. coli infection alone. Finally, we demonstrated that coinoculation of E. faecalis with a commensal strain of E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support the hypothesis that E. faecalis suppression of NF-κB-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis, especially during coinfection with less virulent or commensal E. coli strains.

Classical swine fever virus Shimen infection increases p53 signaling to promote cell cycle arrest in porcine alveolar macrophages.

Classical swine fever virus (CSFV) replicates in macrophages and causes persistent infection. Despite its role in disastrous economic losses in swine industries, the molecular mechanisms underlying its pathogenesis are poorly understood. The virus evades the neutralizing immune response, subverting the immune system to ensure its own survival and persistence. Our genome-wide analysis of porcine alveolar macrophage transcriptional responses to CSFV Shimen infection using the Solexa/Illumina digital gene expression system revealed that p53 pathway components and cell cycle molecules were differentially regulated during infection compared to controls. Further, we investigated the molecular changes in macrophages infected with CSFV Shimen, focusing on the genes involved in the p53 pathway. CSFV Shimen infection led to phosphorylation and accumulation of p53 in a time-dependent manner. Furthermore, CSFV Shimen infection upregulated cyclin-dependent kinase inhibitor 1A (p21) mRNA and protein. In addition, CSFV Shimen infection induced cell cycle arrest at the G1 phase, as well as downregulation of cyclin E1 and cyclin-dependent kinase 2 (CDK2). The expression of genes in the p53 pathway did not change significantly after p53 knockdown by pifithrin-α during CSFV Shimen infection. Our data suggest that CSFV Shimen infection increases expression of host p53 and p21, and inhibits expression of cyclin E1 and CDK2, leading to cell cycle arrest at the G1 phase. CSFV may utilize this strategy to subvert the innate immune response and proliferate in host cells.

Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection.

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

Macrophage cell death and transcriptional response are actively triggered by the fungal virulence factor Cbp1 during H. capsulatum infection.

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis.

Cytosolic Phospholipase A2α and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation

The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α+/+ than cPLA2α-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α+/+ than cPLA2α-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α+/+ and cPLA2α-/- macrophages (3 h) was compared by microarray. cPLA2α+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α-/- macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α+/+ macrophages. Representative genes expressed lower in cPLA2α+/+ macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.

Brucella melitensis, B. neotomaeandB. ovisElicit Common and Distinctive Macrophage Defense Transcriptional Responses

Brucella spp. establish an intracellular replicative niche in macrophages, while macrophages attempt to eliminate the bacteria by innate defense mechanisms. Brucella spp. possess similar genomes yet exhibit different macrophage infections. Few B. melitensis and B. neotomae enter macrophages with intracellular adaptation occurring over 4-8 hr. Conversely, B. ovis are readily ingested by macrophages and exhibit a persistent plateau of infection. Evaluating early macrophage interaction with Brucella spp. allows discovery of host entry and intracellular translocation mechanisms. Microarray analysis of macrophage transcriptional response following a 4 hr infection by different Brucella spp. revealed common macrophage genes altered in expression compared to uninfected macrophages. Macrophage infection with three different Brucella spp. provokes a common innate immune theme with increased transcript levels of chemokines and defense response genes and decreased transcript levels of GTPase signaling and cytoskeletal function that may affect trafficking of Brucella containing vesicles. For example, transcript levels of genes associated with chemotaxis (IL-1beta, MIP-1alpha), cytokine regulation (Socs3) and defense (Fas, Tnf) were increased, while transcript levels of genes associated with vesicular trafficking (Rab3d) and lysosomal associated enzymes (prosaposin) were decreased. Genes with altered macrophage transcript levels among Brucella spp. infections may correlate with species specific host defenses and intracellular survival strategies. Depending on the infecting Brucella species, gene ontology categorization identified genes differentially involved in cell growth and maintenance, endopeptidase inhibitor activity and G-protein mediated signaling. Examples of decreased gene expression in B. melitensis infection but not other Brucella spp. were growth arrest (Gas2), immunoglobulin receptor (FcgammarI) and chemokine receptor (Cxcr4) genes, suggesting opposing effects on intracellular functions.

Distinct TLR- and NLR-Mediated Transcriptional Responses to an Intracellular Pathogen

How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNβ. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNβ was reduced in nod2 −/− macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNβ induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens.

Classical swine fever virus inhibits nitric oxide production in infected macrophages

Classical swine fever virus (CSFV)-macrophage interactions during infection were analysed by examining macrophage transcriptional responses via microarray. Eleven genes had increased mRNA levels (>2.5-fold, P<0.05) in infected cell cultures, including arginase-1, an inhibitor of nitric oxide production, phosphoinositide 3-kinase, chemokine receptor 4 and interleukin-1beta. Lower levels of nitric oxide and increased arginase activity were found in CSFV-infected macrophages. These changes in gene expression in macrophages suggest viral modulation of host expression to suppress nitric oxide production.

Effect of phthiocerol dimycocerosate deficiency on the transcriptional response of human macrophages to Mycobacterium tuberculosis

The control of mycobacterial infections is dependent on the finely tuned synergism between the innate and adaptive immune responses. The macrophage is the major host cell for Mycobacterium tuberculosis and the degree of virulence of mycobacteria may influence the initial macrophage response to infection. The cell wall molecule, phthiocerol dimycocerosate (DIM), is an important virulence factor that influences the early growth of M. tuberculosis in the lungs. To explore the basis for this effect we have compared the early gene response of human THP-1 macrophages to infection with virulent M. tuberculosis and the DIM-deficient ΔfadD26 M. tuberculosis strain using microarrays. Detailed analysis revealed a common core of macrophage genes, which were rapidly induced following infection with both strains, and deficiency of DIM had no significant effect on this initial macrophage transcriptional responses. In addition to chemokines and pro-inflammatory cytokines, the early response genes included components of the Toll-like receptor signalling, antigen presentation and apoptotic pathways, interferon response genes, cell surface receptors and their ligands, including TNF-related apoptosis inducing ligand (TRAIL) and CD40, and other novel genes. Therefore, although fadD26 deficiency is responsible for the early attenuation of the growth of M. tuberculosis in vivo, this effect is not associated with differences in the initial macrophage transcriptional response.

Macrophage Transcriptional Responses following In Vitro Infection with a Highly Virulent African Swine Fever Virus Isolate

We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1beta and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells.