Purinergic signalling in host innate immune defence against intracellular pathogens.

Purinergic signalling is an evolutionarily conserved signalling pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides released from host cells during intracellular pathogen infections activate plasma membrane purinergic type 2 receptors (P2 receptors) that stimulate microbicidal mechanisms in host innate immune cells. P2X ion channels and P2Y G protein-coupled receptors are involved in activating host innate immune defence mechanisms, phagocytosis, phagolysosomal fusion, production of reactive species, acidification of parasitophorous vacuoles, inflammasome activation, and the release of cytokines, chemokines, and other inflammatory mediators. In this review, as part of a special issue in tribute to Geoffrey Burnstock, we discuss advances in understanding the importance of P2 receptors in the host antimicrobial innate mechanisms against intracellular pathogen infections.

Phthiocerol dimycocerosates promote access to the cytosol and intracellular burden of Mycobacterium tuberculosis in lymphatic endothelial cells.

BACKGROUND: Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs. RESULTS: Analysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden. CONCLUSIONS: PDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans.

Dual phosphorylation in response regulator protein PrrA is crucial for intracellular survival of mycobacteria consequent upon transcriptional activation.

The remarkable ability of Mycobacterium tuberculosis (Mtb) to survive inside human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belonging to an essential two-component system (TCS), PrrA/B, which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. In the present study, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein. We have further characterized the role of terminal phospho-acceptor domain in the physical interaction of PrrA with its cognate kinase PrrB. The genetic deletion of prrA/B in Mycobacterium smegmatis was possible only in the presence of ectopic copies of the genes, suggesting the essentiality of this TCS in fast-growing mycobacterial strains as well. The overexpression of phospho-mimetic mutant (T6D) altered the growth of M. smegmatis in an in vitro culture and affected the replication of Mycobacterium bovis BCG in mouse peritoneal macrophages. Interestingly, the Thr6 site was found to be conserved in Mtb complex, whereas it was altered in some fast-growing mycobacterial strains, indicating that this unique phosphorylation might be predominant in employing the regulatory circuit in M. bovis BCG and presumably also in Mtb complex.

Assay Development for Image-Based Quantification of Intracellular Bacterial Replication and Analysis of the Innate Immune Response to Infection.

Severe bacterial infection can lead to inflammation, host tissue damage, and ultimately disseminated septic shock. The mammalian innate immune system responds to microbial infection through the detection of invariant pathogen-associated molecular patterns (PAMPs) by a range of pattern recognition receptors (PRRs) expressed by the host cell. A successful immune response involves tightly coordinated signaling from these receptors, leading to a robust transcriptional response producing cytokines and antimicrobial effectors. While the PRR-expressing phagocytes of the host innate immune system function to contain and degrade internalized bacteria through pathways such as selective autophagy, pathogenic bacteria may subvert this process to replicate in the host cell. We describe the development of imaging assays to investigate these host-pathogen interactions through gene perturbation screens, which could lead to the identification of novel effectors of the host response to bacterial infection. We identify markers of coordinated initial signaling in macrophages challenged with ligands to PRRs of the toll-like receptor (TLR) family and compare this response to that induced by intact bacteria of the Burkholderia cenocepacia complex (Bcc), an opportunistic pathogen that causes life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Bcc has been shown to escape the endocytic pathway, activate selective autophagy, and replicate within human macrophages. We demonstrate robust image-based quantification of multiple stages of Bcc infection of macrophages: ubiquitin tagging of cytosolic bacteria, recruitment of selective autophagy effector proteins, and intracellular bacterial replication, and we show perturbation of bacterial replication using drug treatment or siRNA-based gene knockdown. The described panel of imaging assays can be extended to other bacterial infections and pathogenic ligand combinations where high-content siRNA screening could provide significant new insight into regulation of the innate immune response to infection.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures.

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4 min injection assay to detect total microcystins in water samples below the WHO recommended limit (1 µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5 ng/mL for extracellular toxins and 0.05 ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Intracellular pathogen detection by RIG-I-like receptors.

The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 trigger innate immune responses against viral infections that serve to limit virus replication and to stimulate adaptive immunity. RLRs are cytosolic sensors for virus-derived RNA and thus responsible for intracellular immune surveillance against infection. RLR signaling requires the adapter protein MAVS to induce type I interferon, interferon-stimulated genes, and proinflammatory cytokines. This review focuses on the molecular and cell biological requirements for RLR signal transduction.

Macrophages from elders are more permissive to intracellular multiplication of Mycobacterium tuberculosis.

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-γ, but not IL-1ß, IL-6 or TNF-α, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth.

Antigen-driven induction of polyreactive IgM during intracellular bacterial infection.

Polyreactivity is well known as a property of natural IgM produced by B-1 cells. We demonstrate that polyreactive IgM is also generated during infection of mice with Ehrlichia muris, a tick-borne intracellular bacterial pathogen. The polyreactive IgM bound self and foreign Ags, including single-stranded and double-stranded DNA, insulin, thyroglobulin, LPS, influenza virus, and Borrelia burgdorferi. Production of polyreactive IgM during infection was Ag driven, not due to polyclonal B cell activation, as the majority of polyreactive IgM recognized ehrlichial Ag(s), including an immunodominant outer membrane protein. Monoclonal polyreactive IgM derived from T cell-independent spleen plasmablasts, which was germline-encoded, also bound cytoplasmic and nuclear Ags in HEp-2 cells. Polyreactive IgM protected immunocompromised mice against lethal bacterial challenge infection. Serum from human ehrlichiosis patients also contained polyreactive and self-reactive IgM. We propose that polyreactivity increases IgM efficacy during infection but may also exacerbate or mollify the response to foreign and self Ags.

Counting ion and water molecules in a streaming file through the open-filter structure of the K channel.

The mechanisms underlying the selective permeation of ions through channel molecules are a fundamental issue related to understanding how neurons exert their functions. The "knock-on" mechanism, in which multiple ions in the selectivity filter are hit by an incoming ion, is one of the leading concepts. This mechanism has been supported by crystallographic studies that demonstrated ion distribution in the structure of the Streptomyces lividans (KcsA) potassium channel. These still pictures under equilibrium conditions, however, do not provide a snapshot of the actual, ongoing permeation processes. To understand the dynamics of permeation, we determined the ratio of the ion and water flow [the water-ion coupling ratio (CR(w-i))] through the KcsA channel by measuring the streaming potential (V(stream)) electrophysiologically. The V(stream) value was converted to the CR(w-i) value, which reveals how individual ion and water molecules are queued in the narrow and short filter during permeation. At high K(+) concentrations, the CR(w-i) value was 1.0, indicating that turnover between the alternating ion and water arrays occurs in a single-file manner. At low K(+), the CR(w-i) value was increased to a point over 2.2, suggesting that the filter contained mostly one ion at a time. These average behaviors of permeation were kinetically analyzed for a more detailed understanding of the permeation process. Here, we envisioned the permeation as queues of ion and water molecules and sequential transitions between different patterns of arrays. Under physiological conditions, we predicted that the knock-on mechanism may not be predominant.

The interaction between IL-18 and IL-18 receptor limits the magnitude of protective immunity and enhances pathogenic responses following infection with intracellular bacteria.

The binding of IL-18 to IL-18Rα induces both proinflammatory and protective functions during infection, depending on the context in which it occurs. IL-18 is highly expressed in the liver of wild-type (WT) C57BL/6 mice following lethal infection with highly virulent Ixodes ovatus ehrlichia (IOE), an obligate intracellular bacterium that causes acute fatal toxic shock-like syndrome. In this study, we found that IOE infection of IL-18Rα(-/-) mice resulted in significantly less host cell apoptosis, decreased hepatic leukocyte recruitment, enhanced bacterial clearance, and prolonged survival compared with infected WT mice, suggesting a pathogenic role for IL-18/IL-18Rα in Ehrlichia-induced toxic shock. Although lack of IL-18R decreased the magnitude of IFN-γ producing type-1 immune response, enhanced resistance of IL-18Rα(-/-) mice against Ehrlichia correlated with increased proinflammatory cytokines at sites of infection, decreased systemic IL-10 production, increased frequency of protective NKT cells producing TNF-α and IFN-γ, and decreased frequency of pathogenic TNF-α-producing CD8(+) T cells. Adoptive transfer of immune WT CD8(+) T cells increased bacterial burden in IL-18Rα(-/-) mice following IOE infection. Furthermore, rIL-18 treatment of WT mice infected with mildly virulent Ehrlichia muris impaired bacterial clearance and enhanced liver injury. Finally, lack of IL-18R signal reduced dendritic cell maturation and their TNF-α production, suggesting that IL-18 might promote the adaptive pathogenic immune responses against Ehrlichia by influencing T cell priming functions of dendritic cells. Together, these results suggested that the presence or absence of IL-18R signals governs the pathogenic versus protective immunity in a model of Ehrlichia-induced immunopathology.

Membrane-bound IL-22 after de novo production in tuberculosis and anti-Mycobacterium tuberculosis effector function of IL-22+ CD4+ T cells.

The role of IL-22-producing CD4(+) T cells in intracellular pathogen infections is poorly characterized. IL-22-producing CD4(+) T cells may express some effector molecules on the membrane, and therefore synergize or contribute to antimicrobial effector function. This hypothesis cannot be tested by conventional approaches manipulating a single IL-22 cytokine at genetic and protein levels, and IL-22(+) T cells cannot be purified for evaluation due to secretion nature of cytokines. In this study, we surprisingly found that upon activation, CD4(+) T cells in Mycobacterium tuberculosis-infected macaques or humans could evolve into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Membrane-bound IL-22(+) CD4(+) T effector cells appeared to mature in vivo and sustain membrane distribution in highly inflammatory environments during active M. tuberculosis infection. Near-field scanning optical microscopy/quantum dot-based nanoscale molecular imaging revealed that membrane-bound IL-22, like CD3, distributed in membrane and engaged as ∼100-200 nm nanoclusters or ∼300-600 nm nanodomains for potential interaction with IL-22R. Importantly, purified membrane-bound IL-22(+) CD4(+) T cells inhibited intracellular M. tuberculosis replication in macrophages. Our findings suggest that IL-22-producing T cells can evolve to retain IL-22 on membrane for prolonged IL-22 t(1/2) and to exert efficient cell-cell interaction for anti-M. tuberculosis effector function.

NK cells promote type 1 T cell immunity through modulating the function of dendritic cells during intracellular bacterial infection.

Dendritic cells (DC) play a key role in establishing protective adaptive immunity in intracellular bacterial infections, but the cells influencing DC function in vivo remain unclear. In this study, we investigated the role of NK cells in modulating the function of DC using a murine Chlamydia infection model. We found that the NK cell-depleted mice showed exacerbated disease after respiratory tract Chlamydia muridarum infection, which was correlated with altered T cell cytokine profile. Furthermore, DC from C. muridarum-infected NK-depleted mice (NK(-)DC) exhibited a less mature phenotype compared with that of DC from the infected mice without NK depletion (NK(+)DC). NK(-)DC produced significantly lower levels of both IL-12 and IL-10 than those of NK(+)DC. Moreover, NK(-)DC showed reduced ability to direct primary and established Ag-specific Th1 CD4(+) T cell responses in DC-T coculture systems. More importantly, adoptive transfer of NK(-)DC, in contrast to NK(+)DC, failed to induce type 1 protective immunity in recipients after challenge infection. Finally, NK cells showed strong direct enhancing effect on IL-12 production by DC in an NK-DC coculture system, which was partially reduced by blocking NKG2D receptors signaling and virtually abolished by neutralizing IFN-γ activity. The data demonstrate a critical role of NK cells in modulating DC function in an intracellular bacterial infection.

Cutting edge: primary innate immune cells respond efficiently to polymeric peptidoglycan, but not to peptidoglycan monomers.

The cell wall of bacteria induces proinflammatory cytokines in monocytes and neutrophils in human blood. The nature of the stimulating component of bacterial cell walls is not well understood. We have previously shown polymeric peptidoglycan (PGN) has this activity, and the cytokine response requires PGN internalization and trafficking to lysosomes. In this study, we demonstrate that peptidoglycan monomers such as muramyl dipeptide and soluble peptidoglycan fail to induce robust cytokine production in immune cells, although they activate the nucleotide-binding oligomerization domain proteins in transfected cell models. We further show that lysosomal extracts from immune cells degrade intact peptidoglycan into simpler products and that the lysosomal digestion products activate the nucleotide-binding oligomerization domain proteins. We conclude that naive innate immune cells recognize PGN in its polymeric form rather than monomers such as muramyl dipeptide and require PGN lysosomal hydrolysis to respond. These findings offer new opportunities in the treatment of sepsis, especially sepsis arising from Gram-positive organisms.

Cutting edge: critical role of intracellular osteopontin in antifungal innate immune responses.

We found that absence of osteopontin (OPN) in immunocompromised Rag2(-/-) mice, which lack T and B cells, made the mice extremely susceptible to an opportunistic fungus Pneumocystis, although immunocompetent OPN-deficient mice could clear Pneumocystis as well as wild-type mice. OPN has been studied as an extracellular protein, and the role of an intracellular isoform of OPN (iOPN) is still largely unknown. In this study, we elucidated the mechanism by which iOPN was involved in antifungal innate immunity. First, iOPN was essential for cluster formation of fungal receptors that detect Pneumocystis, including dectin-1, TLR2, and mannose receptor. Second, iOPN played a role as an adaptor molecule in TLR2 and dectin-1 signaling pathways and mediated ERK activation and cytokine production by zymosan, which simultaneously activates TLR2 and dectin-1 pathways. Third, iOPN enhanced phagocytosis and clearance of Pneumocystis. Our study suggests the critical involvement of iOPN in antifungal innate immunity.

Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis.

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells was validated for all 18 epitopes. Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α revealed mono-, dual-, as well as triple-positive CD8(+) T cells, indicating these M. tuberculosis peptide-specific CD8(+) T cells were (poly)functional. Moreover, these T cells were primed during natural infection, because they were absent from M. tuberculosis-noninfected individuals. Control CMV peptide/HLA-A*0201 tetramers stained CD8(+) T cells in M. tuberculosis-infected and noninfected individuals equally, whereas Ebola peptide/HLA-A*0201 tetramers were negative. In conclusion, the M. tuberculosis-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly)functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification.

Infection-induced myelopoiesis during intracellular bacterial infection is critically dependent upon IFN-γ signaling.

Although microbial infections can alter steady-state hematopoiesis, the mechanisms that drive such changes are not well understood. We addressed a role for IFN-γ signaling in infection-induced bone marrow suppression and anemia in a murine model of human monocytic ehrlichiosis, an emerging tick-borne disease. Within the bone marrow of Ehrlichia muris-infected C57BL/6 mice, we observed a reduction in myeloid progenitor cells, as defined both phenotypically and functionally. Infected mice exhibited a concomitant increase in developing myeloid cells within the bone marrow, an increase in the frequency of circulating monocytes, and an increase in splenic myeloid cells. The infection-induced changes in progenitor cell phenotype were critically dependent on IFN-γ, but not IFN-α, signaling. In mice deficient in the IFN-γ signaling pathway, we observed an increase in myeloid progenitor cells and CDllb(lo)Gr1(lo) promyelocytic cells within the bone marrow, as well as reduced frequencies of mature granulocytes and monocytes. Furthermore, E. muris-infected IFN-γR-deficient mice did not exhibit anemia or an increase in circulating monocytes, and they succumbed to infection. Gene transcription studies revealed that IFN-γR-deficient CDllb(lo)Gr1(lo) promyelocytes from E. muris-infected mice exhibited significantly reduced expression of irf-1 and irf-8, both key transcription factors that regulate the differentiation of granulocytes and monocytes. Finally, using mixed bone marrow chimeric mice, we show that IFN-γ-dependent infection-induced myelopoiesis occurs via the direct effect of the cytokine on developing myeloid cells. We propose that, in addition to its many other known roles, IFN-γ acts to control infection by directly promoting the differentiation of myeloid cells that contribute to host defense.

IgM production by bone marrow plasmablasts contributes to long-term protection against intracellular bacterial infection.

IgM responses are well known to occur early postinfection and tend to be short-lived, which has suggested that this Ig does not significantly contribute to long-term immunity. In this study, we demonstrate that chronic infection with the intracellular bacterium Ehrlichia muris elicits a protective, long-term IgM response. Moreover, we identified a population of CD138(high)IgM(high) B cells responsible for Ag-specific IgM production in the bone marrow. The IgM-secreting cells, which exhibited characteristics of both plasmablasts and plasma cells, contributed to protection against fatal ehrlichial challenge. Mice deficient in activation-induced cytidine deaminase, which produce only IgM, were protected against fatal ehrlichial challenge infection. The IgM-secreting cells that we have identified were maintained in the bone marrow in the absence of chronic infection, as antibiotic-treated mice remained protected against challenge infection. Our studies identify a cell population that is responsible for the IgM production in the bone marrow, and they highlight a novel role for IgM in the maintenance of long-term immunity during intracellular bacterial infection.

Internalization and coreceptor expression are critical for TLR2-mediated recognition of lipoteichoic acid in human peripheral blood.

Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.

Impaired germinal center responses and suppression of local IgG production during intracellular bacterial infection.

Germinal centers (GCs) are specialized microenvironments in secondary lymphoid organs that facilitate the development of high-affinity, isotype-switched Abs, and immunological memory; consequently, many infections require GC-derived IgG for pathogen clearance. Although Ehrlichia muris infection elicits a robust expansion of splenic, IgM-secreting plasmablasts, we detected only very low frequencies of isotype-switched IgG-secreting cells in mouse spleens, until at least 3 wk postinfection. Instead, Ag-specific IgG was produced in lymph nodes, where it required CD4 T cell help. Consistent with these findings, organized GCs and phenotypically defined splenic GC B cells were found in lymph nodes, but not spleens. Ehrlichial infection also inhibited spleen IgG responses against a coadministered T cell-dependent Ag, hapten 4-hydroxy-3-nitrophenyl acetyl (NP)-conjugated chicken gamma globulin in alum. NP-specific B cells failed to undergo expansion and differentiation into GC B cells in the spleen, Ab titers were reduced, and splenic IgG production was inhibited nearly 10-fold when the Ag was administered during infection. Our data provide a mechanism whereby an intracellular bacterial infection can compromise local immunity to coinfecting pathogens or antigenic challenge.

Francisella acid phosphatases inactivate the NADPH oxidase in human phagocytes.

Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [DeltaABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the DeltaABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida DeltaABCH strain stimulated a significant level of reactive oxygen species. The DeltaABCH mutant, but not the wild-type strain, strongly colocalized with p47(phox) and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47(phox) knockout mice. Finally, purified AcpA strongly dephosphorylated p47(phox) and p40(phox), but not p67(phox), in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.