Anaplasma phagocytophilum Induces TLR- and MyD88-Dependent Signaling in In Vitro Generated Murine Neutrophils

Abstract

Anaplasma phagocytophilum is a tick-transmitted obligate intracellular Gram-negative bacterium that replicates in neutrophils. It elicits febrile disease in humans and in animals. In a mouse model, elimination of A. phagocytophilum required CD4+ T cells, but was independent of IFN-γ and other classical antibacterial effector mechanisms. Further, mice deficient for immune recognition and signaling via Toll-like receptor (TLR) 2, TLR4 or MyD88 were unimpaired in pathogen control. In contrast, animals lacking adaptor molecules of Nod-like receptors (NLR) such as RIP2 or ASC showed delayed clearance of A. phagocytophilum. In the present study, we investigated the contribution of further pattern recognition receptor (PRR) pathways to the control of A. phagocytophilum in vivo. Mice deficient for the NLR NOD2 had elevated bacterial loads in the early phase of infection, but were unimpaired in pathogen elimination. In contrast, animals lacking adaptor proteins of different C-type lectin receptors (CLR) such as DAP12, Fc-receptor γ-chain (FcRγ) and SYK controlled A. phagocytophilum as efficiently as wild-type mice. Further, we investigated which PRR pathways are involved in the sensing of A. phagocytophilum by in vitro generated Hoxb8 murine neutrophils. In vitro, recognition of A. phagocytophilum by murine neutrophils was dependent on TLR- and MyD88 signaling. However, it remained intact in the absence of the NLR NOD1, NOD2 and NALP3 and of the CLR adaptor molecules DAP12 and FcRγ. From these results, we conclude that TLR rather than NLR or CLR are critical for the detection of A. phagocytophilum by neutrophils although in vivo defective TLR-signaling is compensated probably because of the redundancy of the immune system.

Pattern Recognition Receptors in Innate Immunity to Obligate Intracellular Bacteria

“…Recently, the link between MyD88-or TRIF-dependent TLRs and inflammation has been explored via in vitro and in vivo approaches. Infected MyD88 −/− , MyD88/ TRIF −/− , and TLR2/3/4/7/9 −/− murine Hoxb8 neutrophils, compared with WT cells, show diminished proinflammatory responses (Nos2 transcripts; TNFα, CCL4, and CCL5 secretion) [98]. Despite differences in inflammation in vitro, no phenotypic differences have been observed in vivo between infected WT C57BL/6 and TRIF −/− mice.…”

“…The authors observed significantly increased A. phagocytophilum loads in the blood and lungs of NOD2 −/− C57BL/6 mice throughout the course of infection, but both NOD2 −/− and WT mice eventually cleared the bacterium at similar rates [98]. In experiments using Hoxb8 murine neutrophils, no differences in bacterial load or proinflammatory markers have been observed among infected WT, NOD1 −/− , NOD2 −/− , and NLRP3 −/− cells [98]. However, A. phagocytophilum has been shown to activate NLRC4 via a unique mechanism.…”

“…Vacuole NOD2 [98] RIP2 [98] NLRC4 [100] MyD88 [98] TLR2 [96] STAT1 [96] Ehrlichia chaffeensis White-tailed deer*, coyotes, and dogs Unknown Amblyomma*, Ixodes, and Dermacentor…”

“…Thus, NLRs signaling through RIP2 have been speculated to contribute to mounting of the Th1 response to Anaplasma [99]. A separate study by Müller and colleagues has built upon this hypothesis by analyzing the contributions of specific NLRs during infection [98]. The authors observed significantly increased A. phagocytophilum loads in the blood and lungs of NOD2 −/− C57BL/6 mice throughout the course of infection, but both NOD2 −/− and WT mice eventually cleared the bacterium at similar rates [98].…”

“…A separate study by Müller and colleagues has built upon this hypothesis by analyzing the contributions of specific NLRs during infection [98]. The authors observed significantly increased A. phagocytophilum loads in the blood and lungs of NOD2 −/− C57BL/6 mice throughout the course of infection, but both NOD2 −/− and WT mice eventually cleared the bacterium at similar rates [98]. In experiments using Hoxb8 murine neutrophils, no differences in bacterial load or proinflammatory markers have been observed among infected WT, NOD1 −/− , NOD2 −/− , and NLRP3 −/− cells [98].…”