Anaplasma Phagocytophilum

Maiara S Severo; Kimberly D Stephens; Michail Kotsyfakis; Joao HF Pedra

Disclosures

Future Microbiol. 2012;7(6):719-731. 

In This Article

A. phagocytophilum & humans: just an accident

How does HGA Happen?

Human outdoor activities, especially during the warmer spring and summer months, may lead to tick exposure and A. phagocytophilum infection. If tick feeding and pathogen transmission take place successfully, symptoms such as fever, chills, headache and muscle aches may occur.[4] The underlying events that lead to disease manifestation are not well understood. A remarkable feature of HGA is that this illness does not result from direct pathogen load, but from host-derived immunopathology. Furthermore, neutrophils do not seem to play a major role in A. phagocytophilum immunity, as these cells are not efficient in clearing A. phagocytophilum infection, have their signaling pathways modulated by A. phagocytophilum, and are significantly decreased during infection. Monocytes and macrophages, on the other hand, play a critical role in combating A. phagocytophilum infection. Dumler and colleagues have described that A. phagocytophilum triggers proinflammatory responses in macrophages via NF-κB in primary murine macrophages through the TLR2.[47] Consistent with this, Rikihisa and Kim have also shown that A. phagocytophilum transduces different signals between neutrophils and monocytes for cytokine generation.[48]

Decreased bone marrow function and changes in hematopoietic progenitor and peripheral blood cells in the spleen have been described in acute infection with A. phagocytophilum.[49] This has been associated with aberrant CXCL12/CXCR4 signaling and hematopoietic stem cell mobilization.[50] Studies characterizing cytokine response to A. phagocytophilum infection indicate that the response favors the Th1 phenotype. In addition, cytokine secretion varies according to the type of cell investigated, the sensitivity of the technique adopted during experimentation, and the approach used during pathogen infection. For example, Klein et al. did not report any measurable IL-1, IL-6 or TNF-α secretion in culture supernatants from HL-60 cells or cells enriched for marrow progenitors at any of the time points assayed.[51] Conversely, Johns et al. reported TNF-α and IL-6 secretion after A. phagocytophilum stimulation of bone marrow cells in an ex vivo model system.[49]

In the early phase of infection, IL-12/23p40 regulates CD4+ T cells, while IL-12/23p40-independent mechanisms contribute to pathogen elimination from the host.[52] IL-18 produced by the inflammasome, a protein scaffold associated with the inflammatory process, also regulates CD4+ T-cell responses.[53] Mice vaccinated with A. phagocytophilum or protected by passive immunization also become refractory to A. phagocytophilum infection, suggesting that antibodies may participate in A. phagocytophilum immunity.[54] Expression of Toll-like receptors or myd88 remains unaltered during A. phagocytophilum infection of neutrophils,[55] and JNK2 inhibits production of IFN-γ by NK T cells upon A. phagocytophilum challenge in mice.[56] IFN-γ seems to play a role in controlling A. phagocytophilum infection and immunopathology. In IFN-γ-knockout mice, bacterial levels in the tissues are increased in the early phase of infection, but tissue damage is absent and bacteria are eventually eliminated. The same study described increased lesions in IL-10-knockout mice, which showed normal levels of IFN-γ.[57]

Hepatic histopathology upon infection was dependent on IFN-γ produced by NK, and to a lesser extent, NK T cells.[58] Corroborating these findings, lipoprotein or glycolipid components of A. phagocytophilum membranes stimulate innate immune cell proliferation.[59] In addition, cd1d-knockout mice had a higher pathogen load at day 2 than the wild-type animals. Injection of α-galactosylceramide, a strong NK T-cell agonist, also increased IFN-γ release and protected mice from A. phagocytophilum.[56] Mice deficient in TLR2, TLR4, iNOS, MyD88, TNF and NADPH oxidase have been studied, and they are all capable of clearing A. phagocytophilum infection.[60] However, these innate immune molecules seem important for host-derived immunopathology.[61] CD11b and CD18, on the other hand, are crucial for bacterial clearance because infection of CD11b/CD18-knockout mice leads to an increase in bacterial load when compared with wild type mice.[62] Furthermore, the NLRC4, but not the NLRP3 inflammasome, is partially required for A. phagocytophilum host response in vivo.[53] Taken together, a concerted effort by several research groups have illustrated that A. phagocytophilum immunity is complex and multifactorial.

Downregulation of Oxidative & Inflammatory Responses

Microarray analysis in neutrophils, together with proteomic analysis in HL-60 cells, indicated that genes and proteins involved in innate immunity and inflammation have their expression modulated by A. phagocytophilum infection.[1] Neutrophils are the most abundant type of phagocyte and the major mediator of the respiratory burst activated upon exposure to pathogens. A. phagocytophilum does not carry genes involved in detoxification and does not induce reactive oxygen species when infecting murine or human neutrophils.[1] Moreover, this pathogen inhibits mRNA expression of gp91phox (also known as NOX2) and decreases p22phox protein levels, while leaving other components of this system unaffected in human neutrophils.[1] Once infected with A. phagocytophilum, neutrophils become refractory to stimuli such as LPS and phorbol myristate acetate,[63,64] but this active inhibition is not seen in human monocytes.[65] In fact, A. phagocytophilum is easily killed when exposed to reactive oxygen species and this may explain why this pathogen does not infect circulating monocytes. In HL-60 cells, A. phagocytophilum prevents the assembly of NADPH oxidase subunits, and also downregulates NOX2 and surface protein levels.[1] Downregulation of NOX2 has been associated with the production of AnkA by A. phagocytophilum, which has been shown to bind to the CYBB/NOX2 locus.[20,21] Activation of nuclear cathepsin L and enhanced binding of CDP have also been described during A. phagocytophilum infection of neutrophils.[66] Furthermore, A. phagocytophilum minimizes the release of proinflammatory cytokines in human peripheral blood and HL-60 cells.[51] Inhibition of TNF-α, IL-6 and IL-13 was reported in A. phagocytophilum-infected mast cells,[67] suggesting that mitigation of mast cell activation can also contribute to A. phagocytophilum subversion of host defenses. Chromatin modifications within the host cell have been linked to host gene transcription during A. phagocytophilum infection, and gene expression can also be regulated through histone acetylation. Histone modifying enzymes, such as histone deacetylases, maintain histone modification patterns. The upregulation of histone deacetylases together with the epigenetic silencing of host cell defense genes have been described as a requirement for A. phagocytophilum infection of THP1 cells.[68]

Subversion of Host Apoptosis & Autophagy

Neutrophils generally have a very short half-life, which makes it surprising that A. phagocytophilum would find them suitable to inhabit. To survive in a hostile environment, such as inside neutrophils, intracellular pathogens such as A. phagocytophilum are prompted to interfere with host cell apoptosis. A. phagocytophilum inhibits neutrophil apoptosis long enough to develop the morula.[1]A. phagocytophilum infection upregulates expression of anti-apoptotic blc-2 genes, blocks cell surface Fas clustering during spontaneous neutrophil apoptosis and inhibits cleavage of procaspase 8 and caspase 8 activation.[1] Inhibition of Bax translocation into the mitochondria, in addition to activation of caspase 9 and degradation of XIAP, a caspase inhibitor, have also been reported.[1] As previously mentioned, Ats-1 is secreted by the A. phagocytophilum T4SS and prevents mitochondria from responding to apoptotic signals. Autophagy works in synchrony with the host immune response owing to its role in clearing intracellular infections. A. phagocytophilum inclusions display a range of autophagosome markers and do not mature into autolysosomes. Indeed, A. phagocytophilum infection is favored by treatment with rapamycin, an autophagy inducer, but treatment with 3-methyladenine, which inhibits autophagy, reversibly arrests A. phagocytophilum growth without preventing pathogen internalization. This indicates that A. phagocytophilum infection is aided by subverting autophagy.[42] Taken together, A. phagocytophilum manipulates host cell machinery to induce autophagy and cytoplasmic recycling for its own development.

Activation of Protein Kinases

The function of one A. phagocytophilum sensor kinase (PleC) and one response regulator (PleD) have been recently described during cellular infection.[69] The expression of PleC and PleD was increased during A. phagocytophilum exponential growth and was inhibited prior to extracellular release. Recombinant PleD has diguanylate cyclase activity and generates c-di-GMP. c-di-GMP is essential for A. phagocytophilum colonization of mammalian cells, as the c-di-GMP derivative, 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited A. phagocytophilum infection in HL-60 cells.[69] This work was the first description of a functional c-di-GMP signaling pathway in an obligate intracellular pathogen and it suggests that c-di-GMP may be important not only for A. phagocytophilum morulae formation, but also human infection. A. phagocytophilum infection also activates protein kinase pathways in the mammalian host. The p38 MAP kinase, the PKC, PKA and PTK were deemed important for proinflammatory cytokine secretion in monocytes during A. phagocytophilum stimulation.[48] Conversely, the p38 MAP kinase seems important for apoptosis inhibition during A. phagocytophilum infection of neutrophils, as a p38 MAP kinase antagonist increased neutrophil apoptosis during A. phagocytophilum infection.[70]

Neutrophil apoptosis is also regulated by the PI3K/AKT signaling pathway. Sarkar et al. showed that A. phagocytophilum activates the PI3K/AKT pathway, which in turn regulates the expression of the anti-apoptotic protein Mcl-1.[71] The PI3K/AKT pathway also triggers NF-κB activation during pathogen infection of neutrophils and promotes IL-8 secretion in an autocrine manner. This process seems dependent on the E3 ubiquitin ligase cIAP2, as cIAP2 overexpression was observed during A. phagocytophilum infection of neutrophils. Finally, the ERK pathway is activated by A. phagocytophilum in neutrophils. Fikrig and colleagues used a yeast surrogate model and determined that the virulence molecule named AptA activates Erk1/2 phosphorylation and colocalizes with the intermediate filament protein vimentin.[72] The importance of Erk1/2 phosphorylation for A. phagocytophilum infection was independently confirmed by another group. Xiong and Rikihisa showed that the prenylation inhibitor manumycin A inhibited A. phagocytophilum infection of mammalian cells. Furthermore, manumycin A treatment reduced ERK activation in A. phagocytophilum-infected host cells.[73]

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