query: Helminths interfere with immune system control of macrophages activated by IL-4 favor Mycobacterium tuberculosis replication.

positive: T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis. Autophagy is a recently recognized immune effector mechanism against intracellular pathogens. The role of autophagy in innate immunity has been well established, but the extent of its regulation by the adaptive immune response is less well understood. The T helper 1 (Th1) cell cytokine IFN-gamma induces autophagy in macrophages to eliminate Mycobacterium tuberculosis. Here, we report that Th2 cytokines affect autophagy in macrophages and their ability to control intracellular M. tuberculosis. IL-4 and IL-13 abrogated autophagy and autophagy-mediated killing of intracellular mycobacteria in murine and human macrophages. Inhibition of starvation-induced autophagy by IL-4 and IL-13 was dependent on Akt signaling, whereas the inhibition of IFN-gamma-induced autophagy was Akt independent and signal transducer and activator of transcription 6 (STAT6) dependent. These findings establish a mechanism through which Th1-Th2 polarization differentially affects the immune control of intracellular pathogens.

negative: Th2-mediated host protective immunity to intestinal nematode infections. Despite many years of study, relatively little is known about the effector mechanisms that operate against intestine-dwelling nematodes. Most of the current understanding comes from studies of laboratory model systems in rodents. It is clear that when an intestinal helminth infection takes place the immune system generates a strong Th2-mediated response, which regulates a variety of responses characteristic of helminth infections such as eosinophilia, intestinal mastocytosis and elevated IgE production. The ability to modulate the host's immune response in vivo with cytokine-specific monoclonal antibodies and recombinant cytokines, together with the use of animals with disruption of key genes involved in the immune response, have provided powerful tools with which to dissect the potential effector mechanisms operating. In the absence of a T-cell compartment the host is unable to expel the parasite. If a Th1-dominated response is generated, protective immunity is almost universally compromised. Thus, it it would appear that some aspect of a Th2-mediated response controls effector mechanisms. Although it is clear that for some infections the mast cell appears to be involved in protection, probably through the generation of a non-specific inflammatory response, how these cells become activated remains unclear. Data from infections in transgenic animals suggest that activation is not through the high-affinity receptor for IgE. Such studies also call into doubt the importance of conventional interactions between effector leucocytes and antibody. There is little evidence to support a protective role for eosinophilia in any system. New data also imply that, although interleukin 4 (IL-4) is generally important (and can exert effects independent of an adaptive immune response), it is not always sufficient to mediate protection; other Th2 cytokines (e.g. IL-13) may warrant closer investigation. It is apparent that a number of potential Th2-controlled effector mechanisms (some of which may be particularly important at mucosal surfaces) remain to be explored. Overall, it is likely that worm expulsion is the result of a combination of multiple mechanisms, some of which are more critical to some species of parasite than to others.

negative: Mycobacterium tuberculosis phagosome maturation arrest: selective targeting of PI3P-dependent membrane trafficking. The ability of Mycobacterium tuberculosis to enter host macrophages, and reside in a phagosome, which does not mature into a phagolysosome, is central to the spread of tuberculosis and the associated pandemic involving billions of people worldwide. Tuberculosis can be viewed as a disease with a significant intracellular trafficking and organellar biogenesis component. Current understanding of the block in M. tuberculosis phagosome maturation also sheds light on fundamental aspects of phagolysosome biogenesis. The maturation block involves interference with the recruitment and function of rabs, rab effectors (phosphatidylinositol 3-kinases and tethering molecules such as EEA1), SNAREs (Syntaxin 6 and cellubrevin) and Ca2+/calmodulin signaling. M. tuberculosis analogs of mammalian phosphatidylinositols interfere with these systems and associated processes.

negative: Mycobacterium tuberculosis and the environment within the phagosome. Once across the barrier of the epithelium, macrophages constitute the primary defense against microbial invasion. For most microbes, the acidic, hydrolytically competent environment of the phagolysosome is sufficient to kill them. Despite our understanding of the trafficking events that regulate phagosome maturation, our appreciation of the lumenal environment within the phagosome is only now becoming elucidated through real-time functional assays. The assays quantify pH change, phagosome/lysosome fusion, proteolysis, lipolysis, and beta-galactosidase activity. This information is particularly important for understanding pathogens that successfully parasitize the endosomal/lysosomal continuum. Mycobacterium tuberculosis infects macrophages through arresting the normal maturation process of the phagosome, retaining its vacuole at pH 6.4 with many of the characteristics of an early endosome. Current studies are focusing on the transcriptional response of the bacterium to the changing environment in the macrophage phagosome. Manipulation of these environmental cues, such as preventing the pH drop to pH 6.4 with concanamycin A, abrogates the majority of the transcriptional response in the bacterium, showing that pH is the dominant signal that the bacterium senses and responds to. These approaches represent our ongoing attempts to unravel the discourse that takes place between the pathogen and its host cell.

negative: Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

negative: Human IRGM induces autophagy to eliminate intracellular mycobacteria. Immunity-related p47 guanosine triphosphatases (IRG) play a role in defense against intracellular pathogens. We found that the murine Irgm1 (LRG-47) guanosine triphosphatase induced autophagy and generated large autolysosomal organelles as a mechanism for the elimination of intracellular Mycobacterium tuberculosis. We also identified a function for a human IRG protein in the control of intracellular pathogens and report that the human Irgm1 ortholog, IRGM, plays a role in autophagy and in the reduction of intracellular bacillary load.

negative: TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN− dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

negative: Batf is important for IL-4 expression in T follicular helper cells Apart from T helper (Th)-2 cells, T follicular helper (Tfh) cells are a major class of IL-4-producing T cells, required for regulation of type 2 humoral immunity; however, transcriptional control of IL-4 production in Tfh cells remains mainly unknown. Here, we show that the basic leucine zipper transcription factor ATF-like, Batf is important for IL-4 expression in Tfh cells rather than in canonical Th2 cells. Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus. In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells. Batf deficiency impairs the generation of IL-4-producing Tfh cells that results in protection against allergic asthma. Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.

negative: The challenge of new drug discovery for tuberculosis Tuberculosis (TB) is more prevalent in the world today than at any other time in human history. Mycobacterium tuberculosis, the pathogen responsible for TB, uses diverse strategies to survive in a variety of host lesions and to evade immune surveillance. A key question is how robust are our approaches to discovering new TB drugs, and what measures could be taken to reduce the long and protracted clinical development of new drugs. The emergence of multi-drug-resistant strains of M. tuberculosis makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control. Whatever the strategy used, success will depend largely on our proper understanding of the complex interactions between the pathogen and its human host. In this review, we discuss innovations in TB drug discovery and evolving strategies to bring newer agents more quickly to patients.

negative: Polyphosphate kinase 1, a central node in the stress response network of Mycobacterium tuberculosis, connects the two-component systems MprAB and SenX3-RegX3 and the extracytoplasmic function sigma factor, sigma E. Polyphosphate (poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system (TCS) SenX3-RegX3, the extracytoplasmic function sigma factor sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of Mycobacterium tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system SenX3-RegX3 that responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay. The activity of the ppk1 promoter was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation, supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observation that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive-feedback loops in this signalling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system that, in turn, could be a key element of persistence in M. tuberculosis.

negative: Type 2 cytokines: mechanisms and therapeutic strategies Type 2 immune responses are defined by the cytokines interleukin-4 (IL-4), IL-5, IL-9 and IL-13, which can either be host protective or have pathogenic activity. Type 2 immunity promotes antihelminth immunity, suppresses type 1-driven autoimmune disease, neutralizes toxins, maintains metabolic homeostasis, and regulates wound repair and tissue regeneration pathways following infection or injury. Nevertheless, when type 2 responses are dysregulated, they can become important drivers of disease. Type 2 immunity induces a complex inflammatory response characterized by eosinophils, mast cells, basophils, type 2 innate lymphoid cells, IL-4-and/or IL-13-conditioned macrophages and T helper 2 (TH2) cells, which are crucial to the pathogenesis of many allergic and fibrotic disorders. As chronic type 2 immune responses promote disease, the mechanisms that regulate their maintenance are thought to function as crucial disease modifiers. This Review discusses the many endogenous negative regulatory mechanisms that antagonize type 2 immunity and highlights how therapies that target some of these pathways are being developed to treat type 2-mediated disease.

negative: Liver-resident macrophage necroptosis orchestrates type 1 microbicidal inflammation and type-2-mediated tissue repair during bacterial infection. Kupffer cells, the phagocytes of fetal origin that line the liver sinusoids, are key contributors of host defense against enteroinvasive bacteria. Here, we found that infection by Listeria monocytogenes induced the early necroptotic death of Kupffer cells, which was followed by monocyte recruitment and an anti-bacterial type 1 inflammatory response. Kupffer cell death also triggered a type 2 response that involved the hepatocyte-derived alarmin interleukin-33 (IL-33) and basophil-derived interleukin-4 (IL-4). This led to the alternative activation of the monocyte-derived macrophages recruited to the liver, which thereby replaced ablated Kupffer cells and restored liver homeostasis. Kupffer cell death is therefore a key signal orchestrating type 1 microbicidal inflammation and type-2-mediated liver repair upon infection. This indicates that beyond the classical dichotomy of type 1 and type 2 responses, these responses can develop sequentially in the context of a bacterial infection and act interdependently, orchestrating liver immune responses and return to homeostasis, respectively.

negative: Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

negative: IL-1β, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

negative: The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. IL-4 promotes the differentiation of naive CD4+ T cells into IL-4-producing T helper 2 (Th2) cells. Previous work provided suggestive but not conclusive evidence that the transcription factor c-Maf directed the tissue-specific expression of IL-4. It was not known whether c-Maf controlled the transcription of other Th2 cytokine genes. To elucidate the role of c-Maf in vivo, we examined cytokine production in mice lacking c-Maf (c-maf(-/-)). CD4+ T cells and NK T cells from c-maf(-/-) mice were markedly deficient in IL-4 production. However, the mice produced normal levels of IL-13 and IgE, and, when differentiated in the presence of exogenous IL-4, c-maf(-/-) T cells produced approximately normal levels of other Th2 cytokines. We conclude that c-Maf has a critical and selective function in IL-4 gene transcription in vivo.

negative: Suppressor of cytokine signaling 3 inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages. HIV-1 replication within macrophages of the CNS often results in cognitive and motor impairment, which is known as HIV-associated dementia (HAD) in its most severe form. IFN-beta suppresses viral replication within these cells during early CNS infection, but the effect is transient. HIV-1 eventually overcomes this protective innate immune response to resume replication through an unknown mechanism, initiating the progression toward HAD. In this article, we show that Suppressor of Cytokine Signaling (SOCS)3, a molecular inhibitor of IFN signaling, may allow HIV-1 to evade innate immunity within the CNS. We found that SOCS3 is elevated in an in vivo SIV/macaque model of HAD and that the pattern of expression correlates with recurrence of viral replication and onset of CNS disease. In vitro, the HIV-1 regulatory protein transactivator of transcription induces SOCS3 in human and murine macrophages in a NF-kappaB-dependent manner. SOCS3 expression attenuates the response of macrophages to IFN-beta at proximal levels of pathway activation and downstream antiviral gene expression and consequently overcomes the inhibitory effect of IFN-beta on HIV-1 replication. These studies indicate that SOCS3 expression, induced by stimuli present in the HIV-1-infected brain, such as transactivator of transcription, inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages. This consequence of SOCS3 expression in vitro, supported by a correlation with increased viral load and onset of CNS disease in vivo, suggests that SOCS3 may allow HIV-1 to evade the protective innate immune response within the CNS, allowing the recurrence of viral replication and, ultimately, promoting progression toward HAD.

negative: The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. The differential transcriptional response of Mycobacterium tuberculosis to drugs and growth-inhibitory conditions was monitored to generate a data set of 430 microarray profiles. Unbiased grouping of these profiles independently clustered agents of known mechanism of action accurately and was successful at predicting the mechanism of action of several unknown agents. These predictions were validated biochemically for two agents of previously uncategorized mechanism, pyridoacridones and phenothiazines. Analysis of this data set further revealed 150 underlying clusters of coordinately regulated genes offering the first glimpse at the full metabolic potential of this organism. A signature subset of these gene clusters was sufficient to classify all known agents as to mechanism of action. Transcriptional profiling of both crude and purified natural products can provide critical information on both mechanism and detoxification prior to purification that can be used to guide the drug discovery process. Thus, the transcriptional profile generated by a crude marine natural product recapitulated the mechanistic prediction from the pure active component. The underlying gene clusters further provide fundamental insights into the metabolic response of bacteria to drug-induced stress and provide a rational basis for the selection of critical metabolic targets for screening for new agents with improved activity against this important human pathogen.

negative: Genes required for mycobacterial growth defined by high density mutagenesis. Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

negative: Fra-1 protooncogene regulates IL-6 expression in macrophages and promotes the generation of M2d macrophages The tumor microenvironment (TME) plays a prominent role in the growth of tumor cells. As the major inflammatory component of the TME, M2d macrophages are educated by the TME such that they adopt an immunosuppressive role that promotes tumor metastasis and progression. Fra-1 forms activator protein-1 heterodimers with Jun partners and drives gene transcription. Fra-1 is thought to drastically induce tumorigenesis and progression. However, the functional role of Fra-1 in the generation of M2d macrophages is poorly understood to date. Here, we demonstrate that 4T1 mammary carcinoma cells, when co-cultured with RAW264.7 macrophage cells, skew the RAW264.7 macrophage cell differentiation into M2d macrophages. The 4T1 cells stimulate de novo overexpression of Fra-1 in RAW264.7 cells, and then Fra-1 binds to the interleukin 6 (IL-6) promoter to increase the production of the cytokine IL-6 in RAW264.7 cells. IL-6 acts in an autocrine fashion to skew RAW264.7 macrophage cell differentiation into M2d macrophages. These findings open new insights into how to reverse M2d macrophage-induced immune tolerance to improve the efficacy of immunotherapeutic approaches.

negative: Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus–induced Colitis We have previously demonstrated that interleukin (IL)-10–deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus . Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4+ T cells from IL-10 KO animals and that the cotransfer of CD4+ T cells from H. hepaticus –infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4+ cells are contained within the CD45RBlow fraction and unexpectedly were found in both the CD25+ and the CD25− subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25+ and CD25− CD45RBlow CD4+ cells block colitis involves IL-10 and not transforming growth factor (TGF)-β, as treatment with anti–IL-10R but not anti–TGF-β monoclonal antibody abrogated their protective effect. In vitro, CD45RBlow CD4+ cells from infected WT mice were shown to produce IL-10 and suppress interferon-γ production by IL-10 KO CD4+ cells in an H. hepaticus antigen–specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.

negative: Role of chicken melanoma differentiation-associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 107 plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-β (IFN-β) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2’, 5’-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1β (IL-1β) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 μM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-β and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

negative: Simian immunodeficiency virus–induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor–deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

negative: Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, resides in a distinct vacuole structure called Legionella-containing vacuole (LCV). The LCV resists fusion with the lysosome and permits efficient bacterial replication in host macrophages, which requires a Dot/Icm type IVB secretion system. Dot/Icm-translocated effector SdhA is critical for L. pneumophila intracellular growth and functions to prevent host cell death. Here, we show that the absence of SdhA resulted in elevated caspase-1 activation and IL-1β secretion as well as macrophage pyroptosis during Legionella infection. These inflammasome activation phenotypes were independent of the established flagellin-NAIP5-NLRC4 axis, but relied on the DNA-sensing AIM2 inflammasome. We further demonstrate that Legionella DNA was released into macrophage cytosol, and this effect was significantly exaggerated by the absence of SdhA. SdhA bears a functional Golgi-targeting GRIP domain that is required for preventing AIM2 inflammasome activation. Ectopically expressed SdhA formed a unique ring-shape membrane structure, further indicating a role in membrane trafficking and maintaining LCV membrane integrity. Our data together suggest a possible link, mediated by the function of SdhA, between LCV trafficking/maturation and suppression of host innate immune detection.

negative: Programmed death-1–induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in CD4+ T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible CD4+ T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1.

negative: Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome. Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1β/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1β processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1β expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

negative: Inflammasome-induced IL-1β secretion in microglia is characterized by delayed kinetics and is only partially dependent on inflammatory caspases. Inflammasomes are multiprotein complexes that link pathogen recognition and cellular stress to the processing of the proinflammatory cytokine interleukin-1β (IL-1β). Whereas inflammasome-mediated activation is heavily studied in hematopoietic macrophages and dendritic cells, much less is known about microglia, resident tissue macrophages of the brain that originate from a distinct progenitor. To directly compare inflammasome-mediated activation in different types of macrophages, we isolated primary microglia and hematopoietic macrophages from adult, healthy rhesus macaques. We analyzed the expression profile of NOD (nucleotide-binding oligomerization domain)-like receptors, adaptor proteins, and caspases and characterized inflammasome activation and regulation in detail. We here demonstrate that primary microglia can respond to the same innate stimuli as hematopoietic macrophages. However, microglial responses are more persistent due to lack of negative regulation on pro-IL-1β expression. In addition, we show that while caspase 1, 4, and 5 activation is pivotal for inflammasome-induced IL-1β secretion by hematopoietic macrophages, microglial secretion of IL-1β is only partially dependent on these inflammatory caspases. These results identify key cell type-specific differences that may aid the development of strategies to modulate innate immune responses in the brain.

negative: Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome. The proinflammatory cytokine IL-1beta plays an important role in antifungal immunity; however, the mechanisms by which fungal pathogens trigger IL-1beta secretion are unclear. In this study we show that infection with Candida albicans is sensed by the Nlrp3 inflammasome, resulting in the subsequent release of IL-1beta. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome, as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL- 1beta secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans, which is consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.

negative: The association between sterilizing activity and drug distribution into tuberculosis lesions Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. In contrast, moxifloxacin, which is active in vitro against a subpopulation of Mycobacterium tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug-resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration may contribute to treatment outcome has wide implications for TB.

negative: Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung. The human tuberculous granuloma provides the morphological basis for local immune processes central to the outcome of tuberculosis. Because of the scarcity of information in human patients, the aim of the present study was to gain insights into the functional and structural properties of infiltrated tissue. To this end, the mycobacterial load in lesions and dissemination to different tissue locations were investigated, as well as distribution, biological functions, and interactions of host immune cells. Analysis of early granuloma formation in formerly healthy lung tissue revealed a spatio-temporal sequence of cellular infiltration to sites of mycobacterial infection. A general structure of the developing granuloma was identified, comprising an inner cell layer with few CD8(+) cells surrounding the necrotic centre and an outer area of lymphocyte infiltration harbouring mycobacteria-containing antigen-presenting cells as well as CD4(+), CD8(+), and B cells in active follicle-like centres resembling secondary lymphoid organs. It is concluded that the follicular structures in the peripheral rim of granulomas serve as a morphological substrate for the orchestration of the enduring host response in pulmonary tuberculosis.

negative: Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis. Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2. Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

negative: Disease-associated functions of IL-33: the new kid in the IL-1 family Interleukin-33 (IL-33), a newly described member of the IL-1 family, is expressed by many cell types following pro-inflammatory stimulation and is thought to be released on cell lysis. The IL-33 receptor, consisting of ST2 and IL-1 receptor accessory protein, is also widely expressed, particularly by T helper 2 (TH2) cells and mast cells. IL-33 is host-protective against helminth infection and reduces atherosclerosis by promoting TH2-type immune responses. However, IL-33 can also promote the pathogenesis of asthma by expanding TH2 cells and mediate joint inflammation, atopic dermatitis and anaphylaxis by mast cell activation. Thus IL-33 could be a new target for therapeutic intervention across a range of diseases.

negative: The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells. Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.

negative: IL-6 as a keystone cytokine in health and disease Interleukin 6 (IL-6) has a broad effect on cells of the immune system and those not of the immune system and often displays hormone-like characteristics that affect homeostatic processes. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a prominent target for clinical intervention. However, the signaling cassette that controls the activity of IL-6 is complicated, and distinct intervention strategies can inhibit this pathway. Clinical experience with antagonists of IL-6 has raised new questions about how and when to block this cytokine to improve disease outcome and patient wellbeing. Here we discuss the effect of IL-6 on innate and adaptive immunity and the possible advantages of various antagonists of IL-6 and consider how the immunobiology of IL-6 may inform clinical decisions.