Innate Immune Response to Anaplasma phagocytophilum Contributes to Hepatic Injury

ABSTRACT In mice, Anaplasma phagocytophilum control is independent of phagocyte oxidase (phox), inducible NO synthase (NOS2), tumor necrosis factor (TNF), and MyD88 Toll-like receptor signaling. We show that despite evasion of these host responses, phox, NOS2, TNF, and MyD88 are activated and contribute to inflammation and hepatic injury more than A. phagocytophilum itself.

Anaplasma phagocytophilum is an obligate intraneutrophilic tick-borne bacterium that causes human and animal granulocytic anaplasmosis (HGA). In the murine model of HGA, host immune response is more important for histopathologic lesions than is pathogen load (24,29). Deficiency in inducible nitric oxide synthase (NOS2), tumor necrosis factor (TNF), or NADPH phagocyte oxidase (phox) has little impact on A. phagocytophilum control (5,32). Likewise, infected Toll-like receptor 2 (TLR2)-deficient (Tlr2 Ϫ/Ϫ ), TLR4-deficient, and Myd88 Ϫ/Ϫ mice attain pathogen load levels similar to those of controls (32), despite A. phagocytophilum vs triggering NF-B activation through TLR2 in vitro (17). A. phagocytophiluminfected mice lacking gamma interferon have fewer histopathologic changes and increased pathogen loads, yet gamma interferon is dispensable for pathogen control (2,24). Whether induced immune effectors cause the inflammatory tissue damage underlying disease is unknown. We hypothesized that A. phagocytophilum, although able to avoid innate immunity, nevertheless induces innate immune effectors, resulting in inflammatory hepatic lesions, which are elicited independently of bacterial load.
A. phagocytophilum (strain MRK) was grown in HL-60 cells, and C.B17 SCID mice were infected as described previously (32). For experimental infections, mice were injected intraperitoneally with 100 l of blood that was pooled from infected or uninfected (HL-60 cells only) SCID mice and diluted 1:5 in phosphate-buffered saline. When tested retrospectively, this dilution corresponded to 10 4 to 10 6 A. phagocytophilum genome equivalents; both the method and the inoculum size were within established ranges for mouse models of A. phagocytophilum infection (2,3,20). For each paired wild-type C5JBL/6 (B6)/knockout mouse experiment, the same inoculum size was used.
Animals were sacrificed and tissues fixed, embedded, and cut into 5-m sections for hematoxylin and eosin staining. Two independent reviewers ranked hepatic inflammatory lesions for severity after normalizing for uninfected controls. Severity was assessed so that high rank numbers reflected higher degrees of tissue inflammation and injury severity. Severity was determined by examining (i) the extents of liver injuries (hepatocyte apoptosis, parenchymal necrosis), (ii) the numbers and relative areas of inflammatory lesions (small multifocal infiltrates to extensive focal infiltration), and (iii) the densities and types of inflammatory infiltrates (predominant or mixed granulocytic, mononuclear, or histiocytic). For infected Cybb Ϫ/Ϫ , Nos2 Ϫ/Ϫ , and paired B6 mice, the median rank for uninfected controls was subtracted from that for infected animals to adjust for background, and then samples were reranked to generate group medians. For other knockout mice, histopathologic results were compared to those for uninfected wild-type mice to assure the absence of concurrent infections or aberrant pathological lesions, but no adjustments for genetic background were made. Because differences between knockout and wildtype mice were potentially not normally distributed, we utilized nonparametric Mann-Whitney statistical analysis; P values of Ͻ0.05 were considered significant.
We previously reported that no significant differences in A. phagocytophilum tissue or blood loads between wild-type and knockout mice were detected at any interval (32). In pilot studies (not shown), histopathologic data pooled from multiple time points showed more-severe hepatic injury in infected Cybb Ϫ/Ϫ mice than in Nos2 Ϫ/Ϫ or B6 mice (P Ͻ 0.05), especially at day 43; no differences were noted between infected Nos2 Ϫ/Ϫ and B6 mice. No significant differences were detected between infected Tnf Ϫ/Ϫ mice and B6 controls at any time point, although there was a trend toward more-severe injury in B6 mice at day 7 (P ϭ 0.06).
In confirmatory studies, all B6 and knockout mice had some hepatic parenchymal and perivascular inflammatory lesions (Fig. 1). Histopathologic ranks are shown in Fig. 2. At the earliest time point, B6 mice had more-severe histopathologic changes (P Ͻ 0.05) than Tnf Ϫ/Ϫ mice, a trend also noted at day 7 (P ϭ 0.06). Likewise, B6 mice had higher severity rankings than Nos2 Ϫ/Ϫ or Cybb Ϫ/Ϫ mice on day 7 (P Ͻ 0.05). By day 42, when hepatic granulomatous lesions were observed in infected Cybb Ϫ/Ϫ mice, severity exceeded the baseline observed for uninfected Cybb Ϫ/Ϫ mice (P Ͻ 0.05) and for infected B6 mice (P ϭ 0.14), although not by a statistically significant degree. This increase in hepatic histopathologic severity was noted for Cybb Ϫ/Ϫ mice only at day 42. No differences in histopathology were identified for other tissues (spleen, lymph node, lung, and bone marrow).
These data show that inflammatory and innate immune pathways are activated during A. phagocytophilum infection, even though A. phagocytophilum has evolved mechanisms to avoid killing by activated effectors (14). We previously showed that innate and adaptive immune responses related to TLR2, TLR4, MyD88, phox, NOS2, and TNF are dispensable for the control of A. phagocytophilum (32). This discrepancy raises the question of whether A. phagocytophilum remains unaffected because it downregulates innate immune activation in vivo or whether it is neither susceptible nor accessible to innate immune effectors.
Histopathologic injury in the HGA murine model does not correlate with bacterial load, and the pathogen itself may not directly cause tissue injury (23,24,29). The reduced histopathologic changes in phox-or NOS2-deficient mice suggest that early innate immune responses elicit inflammatory lesions related to reactive oxygen intermediates (ROI) and reactive nitrogen species (11). Some differences between pilot and confirmatory studies reflect the use of pooled data, the lack of early time intervals, and bias caused by the higher level of histopathologic injury severity in Cybb Ϫ/Ϫ mice at day 43 in the pilot study. TNF appears to have only a limited role in the hepatic injury that accompanies A. phagocytophilum infection, since a significant reduction in severity was observed in only one experiment at a single time point. These kinetics imply that inflammation is largely the result of innate and not adaptive immunity, a finding also supported by the severe histopatho- VOL. 13,2006 NOTES 807 logic changes among infected SCID mice (12). A role for innate immunity via TLRs is also suggested by the inhibition of inflammation in MyD88-deficient B6 mice, because MyD88 is critical for signaling by most TLRs (6,31), and A. phagocytophilum is able to induce NF-B activation via TLR2 but not TLR4 (17). However, this hypothesis is contradicted by the similar levels of inflammation for infected MyD88-deficient C3H mice and for TLR2-and TLR4-deficient C3H mice. These data suggest that with A. phagocytophilum infection, inflammatory signaling in C3H mice (i) proceeds via a MyD88independent pathway and (ii) involves alternative TLRs or unidentified ligands differentially expressed between C3H and B6 mice. Inflammatory lesions among Nos2 Ϫ/Ϫ , Tnf Ϫ/Ϫ , and B6 mice resolved with time. More-severe lesions in Cybb Ϫ/Ϫ mice at day 42 were characterized by granulomatous infiltrates similar to those seen in cases of chronic granulomatous disease (28). The prolonged inflammation illustrates that phox contributes to termination of the inflammatory response in accordance with the immunoregulatory properties of ROI (8). Interestingly, A. phagocytophilum downregulates phox expression and activity in HL-60 cells (4,13,15,26) and induces neutrophil interleukin-8 expression, thereby promoting recruitment of new host cells (3,29). Thus, infection of neutrophils by A. phagocytophilum likely delays inflammation resolution, which is aggravated by the complete absence of phox, as seen for Cybb Ϫ/Ϫ mice (8,25,30).
In early A. phagocytophilum infection, NOS2 promotes hepatic inflammation, consistent with our experiments in which NOS activity was inhibited pharmacologically (11). In contrast, Nos2 Ϫ/Ϫ mice infected by intracellular pathogens such as viruses, Francisella tularensis, or Coxiella burnetii often demonstrate more-severe inflammation (1, 10, 21, 22), which can be FIG. 2. Median histopathologic ranks in hepatic tissues from A. phagocytophilum-infected knockout and control mice. High histopathologic rank numbers correspond to higher degrees of tissue inflammation and severity of injuries. On day 7 of infection, B6 mice had significantly higher (P Ͻ 0.05) severity ranks than did matched (A) Nos2 Ϫ/Ϫ and (B) Cybb Ϫ/Ϫ mice. (C) On day 3, wild-type B6 mice had histopathologic lesions significantly more severe (P Ͻ 0.05) than those of matched Tnf Ϫ/Ϫ mice, a trend also noted at day 7 (P ϭ 0.06). (D) Comparison to the B6 background revealed that Myd88 Ϫ/Ϫ mice had histopathologic lesions less severe (P Ͻ 0.05) than those of infected B6 controls at all time points. (E) When an examination on the C3H/HeN background was conducted, no significant differences between Myd88 Ϫ/Ϫ mice and infected C3H/HeN controls were detected at any time point. (F) Additionally, no differences were noted between infected C3H/HeJ Tlr2 Ϫ/Ϫ mice (TLR2/4 defective) and infected C3H/HeN controls at any time point.
attributed to the lack of NO-dependent feedback inhibition or to the high pathogen load levels after loss of reactive nitrogen species effector function (7,8). We conclude that A. phagocytophilum survival mechanisms for intraneutrophilic growth, i.e., modulations of NOS2, ROI, TNF, and interleukin-8, paradoxically enhance inflammation and tissue injury (9,29). This situation is distinct from that for many other intracellular pathogens that impede innate and adaptive immune responses to evade killing (16,18,19,27). Thus, A. phagocytophilum may have evolved novel adaptations for propagation and pathogenesis not utilized by other intracellular pathogens.