CCR2 and CCR7 promote inflammatory monocyte recruitment through synergistic roles during encephalitic virus infection, but the monocyte response in the brain is dependent on the infecting virus

Inflammatory monocytes (iMO) migrate from the bone marrow to the brain during viral encephalitis. For many viruses, including Herpes simplex virus type-1 (HSV), iMO recruitment is dependent on the chemokine receptor CCR2. However, La Crosse virus (LACV) induces iMO recruitment independent of CCR2. Comparison of iMOs from HSV and LACV-infected mice showed higher expression of the g protein-coupled receptor CCR7 in LACV-induced iMOs. CCR2/CCR7 double knockout mice (DKO) had reduced iMO recruitment following LACV infection compared to CCR2 or CCR7 single knockout mice indicating that each receptor regulated iMO recruitment through complimentary roles. Thus, CCR7 is a novel, synergistic pathway to CCR2-induced iMO recruitment during virus infection. Interestingly, unlike HSV-recruited iMOs, LACV-recruited iMOs did not influence disease and had higher expression of proinflammatory and proapoptotic transcripts but reduced mitotic, phagocytic and phagolysosomal pathway transcripts. These findings indicate that virus-specific activation of iMOs may affect their survival, maturation and functional capabilities. RNA was extracted from ½ brains, and inguinal lymph nodes following a Trizol and chloroform protocol. Brain immune cells from 3 HSV and 3 LACV (both 2 females and 1 male) infected mice were prepared and labeled for FACs isolation as described above. Isolated iMOs from were lysed in RLT lysis buffer. The lysate was combined with additional RLT buffer and beta mercaptoethanol (MilliporeSigma, St. Louis, MO) to bring the final volume to 700 µL with 1% BME. Samples were passed through QIAshredder column (Qiagen, Valencia, CA) at 21,000 x g for 2 minutes to homogenize any genomic DNA. RNA was extracted using Qiagen AllPrep DNA/RNA mini columns (Valencia, CA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer using RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA). RNA was quantitated using a fluorescence assay (Quant-it RiboGreen RNA, Thermofisher Scientific, Waltham, MA) on a Tecan Spark multiplate reader (Tecan, Switzerland). Starting with 200 pg of high-quality total RNA for each sample, volumes were concentrated via speed-vacuum to 9.5 ul. Sequencing libraries were constructed following the Takara SMART-Seq v4 PLUS protocol 011820 (Takara, Mountain View, CA) beginning with oligo(dT) priming/cDNA synthesis and following the manufacturers recommendations. Modifications during cDNA amplification included the use of SeqAmp CB PCR Buffer with 14 cycles of PCR. After AmPure XP bead purification (Agencourt Biosciences, Beverly, MA), cDNA quality was visualized and quantity assessed on a BioAnalyzer HS chip (Agilent Technologies, Inc., Santa Clara, CA). Purified cDNA for each sample was normalized to 1.5 ng in 8 ul volume going into library preparation with the Stem-Loop Adaptor mix. Following 15 cycles of library amplification using balanced SMARTer RNA unique-dual index primers from 96U SetA kit 042121 for Illumina sequencing (Takara, Mountain View, CA), individual libraries were cleaned using an 80% vol of AmPure XP beads, visualized on a BioAnalyzer HS chip, and quantified using the Kapa SYBR FAST Universal qPCR kit for Illumina sequencing (Kapa Biosystems, Wilmington, MA) on the CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Each final library was diluted to a final concentration of 1.5 nM and pooled together in equimolar concentrations for sequencing. After an initial MiSeq paired-end 2 X 150 cycle sequencing run was completed to confirm proper index balancing, samples were run on a NextSeq2k instrument using 1000pM of manually-denatured final library pool and sequenced on a P2 flowcell following a paired-end 2 X 100 run with 300 cycle chemistry.