Liver Is a Generative Site for the B Cell Response to Ehrlichia muris.

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Molecular Pathogenesis of Ehrlichia chaffeensis Infection.

Ehrlichia chaffeensis is an obligatory intracellular and cholesterol-dependent bacterium that has evolved special proteins and functions to proliferate inside leukocytes and cause disease. E. chaffeensis has a multigene family of major outer membrane proteins with porin activity and induces infectious entry using its entry-triggering protein to bind the human cell surface protein DNase X. During intracellular replication, three functional pairs of two-component systems are sequentially expressed to regulate metabolism, aggregation, and the development of stress-resistance traits for transmission. A type IV secretion effector of E. chaffeensis blocks mitochondrion-mediated host cell apoptosis. Several type I secretion proteins are secreted at the Ehrlichia-host interface. E. chaffeensis strains induce strikingly variable inflammation in mice. The central role of MyD88, but not Toll-like receptors, suggests that Ehrlichia species have unique inflammatory molecules. A recent report about transient targeted mutagenesis and random transposon mutagenesis suggests that stable targeted knockouts may become feasible in Ehrlichia.

Intracellular proliferation of Anaplasma phagocytophilum is promoted via modulation of endoplasmic reticulum stress signaling in host cells.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

Serologic evidence of rickettsial diseases associated with tick bites in workers of urban veterinary clinics.

BACKGROUND & OBJECTIVES: Rickettsial and other zoonotic diseases are a latent risk for workers of veterinary clinics. The objective of this study was to evaluate the prevalence and the associated risk factors of parasitosis caused by Rhipicephalus sanguineus, and to estimate the seroprevalence of rickettsial diseases in workers of urban veterinary clinics of Juárez city, México. METHODS: The participants of the study were recruited from 63 private veterinary clinics and hospitals. The serological analysis of the blood samples collected was carried out using immunofluorescence assay (IFA). The statistical analysis for prevalences, risk factors, and correlation was performed with the SAS program. RESULTS: In total, 167 veterinary workers were included in the study. The prevalence of tick bites was 40% (67/167), and the risk factors associated with the occurrence of bites included the activities performed in the clinic and the number of labour hours spent per week. About 21% (35/167) of participants were seropositive to R. rickettsii, 28% (47/167) to Ehrlichia chaffeensis, and 24% (40/167) to Anaplasma phagocytophilum. A correlation was observed between: the number of workers in the clinics and the proportion of tick bites (r2 = 0.865); the prevalence of bites and the seropositivity of the participants to at least one pathogen (r2 = 0.924); and the number of bites per individual and infection to pathogens (r2 = 0.838). INTERPRETATION & CONCLUSION: Workers in urban veterinary clinics are highly exposed to tick bites and, therefore, to the diseases they transmit. Hence, it is important to implement prevention measures and perform constant monitoring of these diseases.

MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1ß and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

Identification of novel immunoreactive proteins and delineation of a specific epitope of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA), an increasingly recognized febrile tick-borne illness, is caused by a gram-negative obligate intracellular bacterium Anaplasma phagocytophilum. Because of nonspecific clinical manifestations, diagnosis of HGA highly depends on laboratory tests. Identification of immunoreactive proteins is prerequisite for development of specific and sensitive immunoassays for HGA. In this study, we identified novel immunoreactive proteins of A. phagocytophilum. Previous studies indicated that secreted proteins of A. phagocytophilum and other bacteria can be immunoreactive antigens. Here we in silico screened A. phagocytophilum genome for encoding proteins which bear features of type IV secretion system substrates. Among seventy seven predicted proteins, fourteen proteins were determined for antigenicity and nine proteins were showed to be immunoreactive antigens. In addition, an APH1384 peptide harboring a B cell epitope predicted by bioinformatics was found specifically reacting with anti-A. phagocytophilum sera. Hereby, we identified novel immunoreactive proteins and delineated a specific epitope of A. phagocytophilum, which might be employed for HGA diagnosis.

Serological reactivity to Anaplasma phagocytophilum in neoehrlichiosis patients.

The tick-borne bacterium Candidatus (Ca.) Neoehrlichia (N.) mikurensis is a cause of "fever of unknown origin" because this strict intracellular pathogen escapes detection by routine blood cultures. Case reports suggest that neoehrlichiosis patients may display serological reactivity to Anaplasma (A.) phagocytophilum. Since Anaplasma serology is part of the diagnostic work-up of undetermined fever in European tick-exposed patients, we wanted to investigate (1) the prevalence of A. phagocytophilum seropositivity among neoehrlichiosis patients, (2) the frequency of misdiagnosed neoehrlichiosis patients among A. phagocytophilum seropositive patients, and (3) the frequency of A. phagocytophilum and Ca. N. mikurensis co-infections. Neoehrlichiosis patients (n = 18) were analyzed for A. phagocytophilum IgM and IgG serum antibodies by indirect immunofluorescence assay. Serum samples from suspected anaplasmosis patients (n = 101) were analyzed for bacterial DNA contents by singleplex PCR specific for A. phagocytophilum and Ca. N. mikurensis, respectively. One fifth of the neoehrlichiosis patients (4/18) were seropositive for IgM and/or IgG to A. phagocytophilum at the time of diagnosis. Among the patients with suspected anaplasmosis, 2% (2/101) were positive for Ca. N. mikurensis by PCR whereas none (0/101) had detectable A. phagocytophilum DNA in the serum. To conclude, patients with suspected anaplasmosis may in fact have neoehrlichiosis. We found no evidence of A. phagocytophilum and Ca. N. mikurensis co-infections in humans with suspected anaplasmosis or confirmed neoehrlichiosis.

Graft loss attributed to possible transfusion-transmitted ehrlichiosis following cord blood stem cell transplant.

We present a case of possible transfusion-transmitted Ehrlichia chaffeensis infection in a heavily transfused cord blood transplant recipient, resulting in severe infection and graft loss. Transfusion-transmitted, vector-borne infections in immunocompromised individuals can have severe consequences, and should be considered in hospitalized patients receiving blood products with unexplained fever or sepsis.

The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

Type I interferon contributes to noncanonical inflammasome activation, mediates immunopathology, and impairs protective immunity during fatal infection with lipopolysaccharide-negative ehrlichiae.

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1ß, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1(-/-) mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3(-/-) mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1(-/-) mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1(-/-) mice also had improved protective immune responses mediated by IFN-γ and CD4(+) Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1(-/-) mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1ß, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4(+) T cells and natural killer T-cell responses against ehrlichiae.

Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival.

Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of ß-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, ß-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.

The omentum is a site of protective IgM production during intracellular bacterial infection.

Infection of mice with the bacterium Ehrlichia muris elicits a protective T cell-independent (TI) IgM response mediated primarily by a population of CD11c-expressing plasmablasts in the spleen. Although splenic marginal zone (MZ) B cells are considered to be important for TI responses to blood-borne pathogens, MZ B cells were not responsible for generating plasmablasts in response to Ehrlichia muris. Moreover, antigen-specific serum IgM was decreased only modestly in splenectomized mice and in mice that lacked spleen, lymph nodes, and Peyer's patches (SLP mice). Both splenectomized and SLP mice were protected against lethal ehrlichial challenge infection. Moreover, we found a high frequency of Ehrlichia-specific plasmablasts in the omentum of both conventional and SLP mice. Omental plasmablasts elicited during Ehrlichia infection lacked expression of CD138 but expressed CD11c in a manner similar to that of their splenic counterparts. Selective ablation of CD11c-expressing B cells nearly eliminated the omental Ehrlichia-specific plasmablasts and reduced antigen-specific serum IgM, identifying the omental B cells as a source of IgM production in the SLP mice. Generation of the omental plasmablasts was route dependent, as they were detected following peritoneal infection but not following intravenous infection. Our data identify the omentum as an important auxiliary site of IgM production during intracellular bacterial infection.

Attenuated Mutants of Ehrlichia chaffeensis Induce Protection against Wild-Type Infection Challenge in the Reservoir Host and in an Incidental Host.

Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease human monocytic ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purified E. chaffeensis mutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-type E. chaffeensis 1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogen's clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations in E. chaffeensis that cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people against Ehrlichia species infections.

Varying influences of selection and demography in host-adapted populations of the tick-transmitted bacterium, Anaplasma phagocytophilum.

BACKGROUND: The host range of a pathogenic bacterial strain likely influences its effective population size, which in turn affects the efficacy of selection. Transmission between competent hosts may occur more frequently for host generalists than for specialists. This could allow higher bacterial population densities to persist within an ecological community and increase the efficacy of selection in these populations. Conversely, specialist strains may be better adapted to their hosts and consequently achieve greater within-host population densities, with corresponding increases in selection efficacy. To assess these different hypotheses, we examined the effective population sizes of three strains of the bacterium Anaplasma phagocytophilum and categorized the varying roles of selection and demography on patterns of genetic diversity and divergence in these populations. A. phagocytophilum is a tick-transmitted, obligately intracellular pathogen. Strains of A. phagocytophilum display varying degrees of host specialization, making this a good species for exploring questions regarding host range, effective population size and selection efficacy. RESULTS: We found that a roe deer specialist harbored the most genetic diversity of the three A. phagocytophilum strains and correspondingly had the largest effective population size. Another strain that is ecologically specialized on rodents and insectivores had the smallest effective population size. However, these mammalian hosts are distantly related evolutionarily. The third strain, a host generalist, was intermediate in its effective population size between the other two strains. Evolutionary constraint on non-synonymous sites was pervasive in all three strains, although some slightly deleterious mutations may also be segregating in these populations. We additionally found evidence of genome-wide selective sweeps in the generalist strain, whereas signals of repeated bottlenecks were detected in the strain with the smallest effective population size. CONCLUSIONS: A. phagocytophilum is a diverse bacterial species that differs among distinct strains in its effective population size, as well as how genetic diversity and divergence have been influenced by selection and demographic changes. In this species, host specialization may facilitate increased population growth and allow more opportunities for selection to act. These results provide insights into how host range has influenced evolutionary patterns of strain divergence in an emerging zoonotic bacterium.

Anaplasma phagocytophilum surface protein AipA mediates invasion of mammalian host cells.

Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick-transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non-phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, we report the significance of a novel A. phagocytophilum invasion protein, AipA. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non-infectious reticulate cell morphotype to its infectious dense-cored morphotype during infection of HL-60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface-exposed, only residues 1 to 87 (AipA1-87 ) were found to be essential for host cell invasion. Recombinant AipA1-87 protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA1-87 , but not other AipA regions, antagonized infection. Additional blocking experiments using peptide-specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA1-87 together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis.

T cell-dependent IgM memory B cells generated during bacterial infection are required for IgG responses to antigen challenge.

Immunological memory has long considered to be harbored in B cells that express high-affinity class-switched IgG. IgM-positive memory B cells can also be generated following immunization, although their physiological role has been unclear. In this study, we show that bacterial infection elicited a relatively large population of IgM memory B cells that were uniquely identified by their surface expression of CD11c, CD73, and programmed death-ligand 2. The cells lacked expression of cell surface markers typically expressed by germinal center B cells, were CD138 negative, and did not secrete Ab ex vivo. The population was also largely quiescent and accumulated somatic mutations. The IgM memory B cells were located in the region of the splenic marginal zone and were not detected in blood or other secondary lymphoid organs. Generation of the memory cells was CD4 T cell dependent and required IL-21R signaling. In vivo depletion of the IgM memory B cells abrogated the IgG recall responses to specific Ag challenge, demonstrating that the cell population was required for humoral memory, and underwent class-switch recombination following Ag encounter. Our findings demonstrate that T cell-dependent IgM memory B cells can be elicited at high frequency and can play an important role in maintaining long-term immunity during bacterial infection.

MyD88 signaling in CD4 T cells promotes IFN-γ production and hematopoietic progenitor cell expansion in response to intracellular bacterial infection.

Hematopoietic stem and progenitor cell (HSPC) phenotype and function can change in response to infectious challenge. These changes can be mediated by cytokines, IFNs, and pathogen-associated molecules, via TLR, and are thought to promote tailored immune responses for particular pathogens. In this study, we investigated the signals that activate HSPCs during ehrlichiosis, a disease characterized by profound hematopoietic dysfunction in both humans and mice. In a mouse model of ehrlichiosis, we observed that infection-induced proliferation of bone marrow HSPCs was dependent on IFN-γ signaling and was partially dependent on MyD88. However, MyD88 was not required in HSPCs for their expansion during infection, because similar frequencies of MyD88-deficient and wild-type HSPCs proliferated in mixed bone marrow chimeric mice. MyD88-deficient mice exhibited low serum and bone marrow concentration of IFN-γ compared with wild-type mice. We next identified CD4 T cells as the primary cells producing IFN-γ in the bone marrow and demonstrated a nonredundant role for CD4-derived IFN-γ in increased HSPCs. Using mixed bone marrow chimeric mice, we identified a requirement for MyD88 in CD4 T cells for increased T-bet expression, optimal IFN-γ production, and CD4 T cell proliferation. Our data demonstrate an essential role for CD4 T cells in mediating HSPC activation in response to bacterial infection and illustrate a novel role for MyD88 signaling in CD4 T cells in this process. These findings further support the idea that IFN-γ production is essential for HSPC activation and hematopoietic responses to infection.

[Monoinfections caused by Borrelia burgdorferi and Borrelia burgdorferi / Anaplasma phagocytophilum co-infections in forestry workers and farmers]. / Monoinfekcja Borrelia burgdorferi i wspólzakazenia Borrelia burgdorferi / Anaplasma phagocytophilum u pracowników lesnictwa i rolników.

BACKGROUND: The presence of co-infections induced by tick-borne pathogens in humans is an important epidemiological phenomenon. This issue has attracted growing attention of doctors and people working under conditions of an increased risk of being exposed to tick bites. MATERIAL AND METHODS: The research group consisted of 93 individuals with current anti-immunoglobulin M/G (IgM/ IgG) Borrelia burgdorferi or IgG anti-Anaplasma phagocytophilum. The respondents were identified during the screening survey in a group of farmers and foresters occupationally exposed to tick bites. The aim of the work was to analyse the frequency of antibodies to specific antigens of B. burgdorferi and the levels of cytokines in forestry workers and farmers with B. burgdorferi monoinfections and B. burgdorferi / A. phagocytophilum co-infections. Statistical analysis was performed using the Chi2, Mann-Whitney U and Kruskal-Wallis tests. RESULTS: There is a stronger generation of IgG antibodies to B. burgdorferi antigens in patients with B. burgdorferi / A. phagocytophilum co-infections, such as variable major protein-like sequence expressed (VlsE) (p < 0.05), p19 (p < 0.02), p17 (p < 0.05) and complement regulator-acquiring surface protein 3 (CRASP3) (p < 0.02) compared to persons with B. burgdorferi monoinfections. The discrepancies in the synthesis of cytokines interleukin 6 (IL-6), IL-10, and tumor necrosis factor α (TNF-α) have not been found in persons with B. burgdorferi monoinfections and B. burgdorferi / A. phagocytophilum co-infection. CONCLUSIONS: The immune response directed against B. burgdorferi is stronger in patients co-infected with B. burgdorferi and A. phagocytophilum than in those with monoinfection.