Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2−/− mice following infection. Interestingly, CCR2 was required for iMO trafficking from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain but was essential for trafficking within the brain parenchyma. Analysis of other orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2 requirement for iMO egress to the blood is not universal for all viruses.

Recruitment of classically activated inflammatory monocytes (iMOs; defined as CCR2+, CD45+, CD11b+, and Ly6Chi) into tissues is a hallmark of inflammation during viral infections. iMOs originate from the bone marrow (BM) and egress to the blood prior to entering infected tissues where they further differentiate into tissue macrophages (1). iMOs are crucial for host defense (2); however, they can also promote damage to the CNS (3, 4). Thus, determining the mechanisms controlling iMO recruitment during encephalitic disease is important for understanding pathogenesis and developing therapeutics.

iMO recruitment from BM to blood is mediated by the chemokine CCL2 and its receptor, CCR2 (1, 57). CCL2 is produced at very low basal levels but is highly upregulated during viral encephalitis (2, 8). CCL2 binds to CCR2 on iMOs, causing desensitization of CXCL12–CXCR4 signaling (9) and leading to decreased expression of the adhesion molecule VLA-4, thus allowing iMO egress from BM to blood (10). Inhibition of CCR2 stops iMOs from entering the blood or inflamed tissues (3, 11). CCR2-deficient mice lack normal levels of iMOs in the blood and do not show recruitment of iMOs in response to West Nile Virus (WNV), HSV-1, or experimental autoimmune encephalitis (2, 12).

La Crosse virus (LACV) belongs to the California serogroup of orthobunyaviruses and is a primary cause of pediatric encephalitis in the United States (13). In humans and mice, LACV disease is associated with infection of neurons and recruitment of leukocytes in the brain. Immunohistochemistry (IHC) showed monocyte/macrophage lineage cells in areas of virus infection in the brain, suggesting that these cells may contribute to pathogenesis (14). To understand the role of iMOs in LACV disease, we analyzed iMO recruitment and migration to the CNS. We found significant iMO recruitment to the brain during LACV disease. Surprisingly, these iMOs were recruited to the blood independently of CCR2. Analysis of related viruses, including Jamestown Canyon virus (JCV) and Tahyna virus (TAHV), showed that these viruses have different mechanisms of monocyte recruitment.

LACV, JCV, and TAHV were described previously (1416). LACV and McKrae HSV-1 (William Halford) stocks were made using Vero cells, whereas JCV, TAHV, and WNV NY99 stocks were made using C636 cells (American Type Culture Collection) and titered using a plaque assay (17).

Studies were completed under National Institutes of Health/Rocky Mountain Laboratories Institutional Animal Care and Use Committee–approved protocols 2012-047 and 2015-023. Ccr2R/R, Macrophage Fas-Induced Apoptosis (MaFIA), and C57BL/6 mice were purchased from The Jackson Laboratory and maintained in breeding colonies at Rocky Mountain Laboratories. Ccr2+/R mice were F1 progeny of Ccr2R/R and C57BL/6 crosses. Three-week-old male and female mice were inoculated i.p. with 103 PFU LACV, JCV, or TAHV or 107 PFU HSV-1 or s.c. with 102 PFU WNV. Supernatant from Vero or C636 cells were used for mock controls. Mice were observed daily for neurologic signs, as previously described (18). For generation of chimeric mice, 7-d-old MaFIA mice were exposed to 200 rad and then given 107 fetal liver cells i.p. from Ccr2R/R mice. After 2 wk, mice were infected with virus.

Cells were isolated from brain and spleen, as previously described (18), or from whole blood and were analyzed by flow cytometry. Fc receptors were blocked using CD16/CD32 FcγIII/II (clone 2.4G2; BD Biosciences). Cells were stained with a combination of CD45 (30-F11), CD11b (M1/70), Ly6C (HK1.4), Ly6G (1A8), CD11c (HL3), pDCA1 (JF05-1C2.4.1), CD8 (53-6.7), CD4 (RM4-5), CD3e (17A2), and F480 (BM8). Cells were analyzed on an LSR II (BD Biosciences), and data were analyzed using FCS Express software (De Novo Software). iMOs were gated as CD45hi, CD11b+, CD11c Ly6G, Ly6Chi (Supplemental Fig. 1). For the CNS, iMOs are expressed as a percentage of all cells isolated by Percoll gradient. For blood analysis, iMOs are expressed as a percentage of CD45hi cells.

IHC of frozen brain tissue was performed, as previously described (19), using polyclonal mouse anti-LACV or rabbit anti-CD31 (Abcam) and donkey anti-rabbit Alexa Fluor 488 or Alexa Fluor 647 and goat anti-mouse Alexa Fluor 488 or Alexa Fluor 594 (Life Sciences). Slides were coverslipped with ProLong Gold Antifade Mountant with DAPI (Life Sciences). In situ hybridization (ISH) of formalin-fixed, paraffin-embedded tissue was performed, as previously described (20), using digoxigenin-labeled RNA sense riboprobes targeting MCP-1 (Ccl2), which were visualized with anti-digoxigenin Ab (Roche) and Fast Red (Dako). Sections were labeled with anti-glial fibrillary acidic protein (GFAP) Abs (Dako) and diaminobenzidine substrate and counterstained with hematoxylin.

Analysis of inflammatory cells in the CNS of LACV-infected mice at the onset of clinical signs revealed Ly6Chi iMOs as the primary immune cell infiltrating into the CNS (Fig. 1A, Supplemental Fig. 1). CD11b+, Ly6Clo macrophage/alternatively activated MOs were the second most abundant cell population, with minor populations of other leukocytes. Time-course analysis showed that iMOs enter the brain by 5 d postinfection (dpi), prior to the onset of clinical disease at 6–7 dpi (Fig. 1B), suggesting that they may have a role in LACV neurologic disease.

FIGURE 1.

iMO migration from BM to blood and CNS is CCR2 independent following LACV infection. (A) Composition of CD45hi Percoll-isolated cells from mouse brains at 7 dpi. (B) Time-course analysis of iMOs in the CNS. Representative flow cytometry data of CD11b+/CD45+ gates from blood (C and D) and brain (E and F) of LACV-infected Ccr2+/R and Ccr2R/R mice at 7 dpi. iMOs are shown in red. (G) Percentage of iMOs in the blood and brain of Ccr2+/R and Ccr2R/R mice at 7 dpi. one-way ANOVA with a Tukey multiple-comparison test. Time course of iMOs in blood (H) and brain (I) of Ccr2+/R and Ccr2R/R mice. For all experiments, n = 2–4 mice for mock-infected groups and n = 6–8 mice for LACV groups per time point. Error bars are SE for all groups. ***p < 0.001, *p < 0.05, two-way ANOVA with a Sidak multiple-comparison test (A, B, H, and I), one-way ANOVA with a Tukey multiple-comparison test (G). n.s., not significant.

FIGURE 1.

iMO migration from BM to blood and CNS is CCR2 independent following LACV infection. (A) Composition of CD45hi Percoll-isolated cells from mouse brains at 7 dpi. (B) Time-course analysis of iMOs in the CNS. Representative flow cytometry data of CD11b+/CD45+ gates from blood (C and D) and brain (E and F) of LACV-infected Ccr2+/R and Ccr2R/R mice at 7 dpi. iMOs are shown in red. (G) Percentage of iMOs in the blood and brain of Ccr2+/R and Ccr2R/R mice at 7 dpi. one-way ANOVA with a Tukey multiple-comparison test. Time course of iMOs in blood (H) and brain (I) of Ccr2+/R and Ccr2R/R mice. For all experiments, n = 2–4 mice for mock-infected groups and n = 6–8 mice for LACV groups per time point. Error bars are SE for all groups. ***p < 0.001, *p < 0.05, two-way ANOVA with a Sidak multiple-comparison test (A, B, H, and I), one-way ANOVA with a Tukey multiple-comparison test (G). n.s., not significant.

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Recruitment of iMOs from the BM to the blood and CNS has been shown to be dependent on CCR2, and mice deficient in Ccr2 are monocytopenic (2). To study iMO recruitment during LACV infection, we used mice containing a red fluorescent protein (RFP) cassette inserted into the open reading frame of the Ccr2 gene, resulting in Ccr2-deficient (Ccr2R/R) mice expressing RFP in place of CCR2 (21). Heterozygous mice, retaining one functional copy of Ccr2 while also expressing RFP (Ccr2+/R), were used as controls. Surprisingly, Ccr2R/R and Ccr2+/R mice infected with LACV had similar numbers of iMOs in their blood (Fig. 1C, 1D, 1G) and brain (Fig. 1E–G) at 6–7 dpi, suggesting that iMO egress to the blood was not dependent on CCR2. To determine when iMOs were recruited to the blood during LACV infection, we examined preclinical time points, 3–5 dpi. Following LACV infection, iMOs in the blood of CCR2R/R mice went from being monocytopenic, as observed in mock-infected animals, to having similar iMOs levels as Ccr2+/R mice by 3–5 dpi in the blood (Fig. 1H) and brain (Fig. 1I). Experiments with unrelated Ccr2−/− mice yielded similar results (Supplemental Fig. 2). Thus, CCR2 was not essential for iMO egress to the blood during LACV infection, which is in direct contrast to the findings for WNV and HSV-1 infections or experimental autoimmune encephalitis (2, 12, 21). To our knowledge, this is the first demonstration that iMO recruitment from the BM to the blood is largely CCR2 independent, and it suggests alternative mechanisms by which iMOs can be recruited from the BM in large quantities during infection. Understanding the mechanisms behind this response would greatly improve our knowledge about iMO development, as well as be a potential therapeutic target to regulate inflammatory responses, either to limit monocyte efflux or to induce large numbers of monocytes.

To ensure that the CCR2-independent recruitment for LACV was not due to alterations in mouse lineage or in gating strategies, we verified CCR2-dependent iMO responses during WNV and HSV-1 infections (Fig. 2A, 2B). To determine whether other orthobunyaviruses related to LACV also induced CCR2-independent iMOs, we infected Ccr2R/R mice with JCV or TAHV. Both viruses produce a detectable infection in mice, although only TAHV was shown to be neurovirulent (15). Interestingly, iMO frequency in blood was CCR2 independent for JCV but not for TAHV infection (Fig. 2C, 2D), including a decrease in iMOs in the brains of TAHV-infected Ccr2R/R mice (Fig. 2E). Thus, CCR2-dependent iMO egress from the BM to the blood varied among orthobunyaviruses, was not dependent on neurovirulence, and varied even between viruses in the same family. Understanding how these viruses induce iMO responses through distinct mechanisms will be important for understanding differences in viral pathogenesis, as well as for determining the basic mechanisms by which iMOs can be recruited during pathogenic infections.

FIGURE 2.

Dependence of CCR2 on iMO egress to blood is pathogen specific. iMOs in the blood of Ccr2+/R and Ccr2R/R mice following infection with HSV-1 at 7 dpi (A) or following infection with WNV at 9–13 dpi (B). Each point represents an individual mouse. *p < 0.05, one-way ANOVA with a Tukey multiple-comparison test, ***p < 0.001, unpaired t test. iMOs in the blood of Ccr2+/R and Ccr2R/R mice shown as the percentage of all CD45+ cells following infection with JCV (C) or TAHV (D). n = 3–8 animals per group at each time point. ***p < 0.001, *p < 0.05, two-way ANOVA analysis with a Sidak multiple-comparison test. (E) iMOs in brains of individual TAHV-infected Ccr2+/R and Ccr2R/R mice with clinical disease. Error bars are SE for all graphs.

FIGURE 2.

Dependence of CCR2 on iMO egress to blood is pathogen specific. iMOs in the blood of Ccr2+/R and Ccr2R/R mice following infection with HSV-1 at 7 dpi (A) or following infection with WNV at 9–13 dpi (B). Each point represents an individual mouse. *p < 0.05, one-way ANOVA with a Tukey multiple-comparison test, ***p < 0.001, unpaired t test. iMOs in the blood of Ccr2+/R and Ccr2R/R mice shown as the percentage of all CD45+ cells following infection with JCV (C) or TAHV (D). n = 3–8 animals per group at each time point. ***p < 0.001, *p < 0.05, two-way ANOVA analysis with a Sidak multiple-comparison test. (E) iMOs in brains of individual TAHV-infected Ccr2+/R and Ccr2R/R mice with clinical disease. Error bars are SE for all graphs.

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Because iMOs may proliferate in the spleen, we analyzed iMOs in the spleen of mock-infected and LACV-infected mice by IHC and flow cytometry. Infection did not induce focal or overall percentage increases in iMOs in the spleen (Fig. 3A–C), suggesting that the spleen was not the source of virus-induced iMOs. To directly confirm that LACV-induced iMOs were not being derived from tissue-resident myeloid cells, we generated chimeric mice in which BM-derived iMOs could be distinguished from resident tissue cells. In short, fetal liver cells from Ccr2R/R mice were used to repopulate partially irradiated MaFIA mice, which express GFP under the CD115 promoter. In these chimeric mice, myeloid cells, including tissue-resident cells, would be GFP positive, whereas cells coming from the BM would be CCR2+ GFP+ or CCR2 RFP+ (Fig. 3D). In mock-infected mice, only 16% of iMOs in the blood were CCR2, correlating with the inability of CCR2 iMOs to egress from the BM (Fig. 3E). However, during LACV infection, the proportion of RFP+ CCR2 cells in the blood increased substantially (Fig. 3F), indicating that BM progenitors, and not tissue-resident cells, were the primary source of iMOs.

FIGURE 3.

LACV-induced CCR2-deficient iMOs are derived from BM. iMOs in the spleen of mock-infected (A) and LACV-infected (B) Ccr2R/R mice at 7 dpi showing a similar staining of RFP+ iMOs (magenta) and cell nuclei (blue) around spleen follicles. Scale bar, 50 μm. (C) Time course of iMOs in spleen during LACV infection in WT mice. (D) Illustration of transfer of Ccr2R/R fetal liver cells to irradiated MaFIA mice. Representative flow data for Ccr2+/+ (GFP) and Ccr2R/R (RFP) iMOs in blood of mock-infected (E) and LACV-infected (F) chimeric mice at 7 dpi. (G and H) IHC image of CCR2+ (GFP) and CCR2 (RFP) iMOs in the brain with staining for CD31+ BVs (white; asterisk) and LACV-infected neurons (blue). Arrows indicate GFP+ cells in close proximity to virus-infected neurons (blue).

FIGURE 3.

LACV-induced CCR2-deficient iMOs are derived from BM. iMOs in the spleen of mock-infected (A) and LACV-infected (B) Ccr2R/R mice at 7 dpi showing a similar staining of RFP+ iMOs (magenta) and cell nuclei (blue) around spleen follicles. Scale bar, 50 μm. (C) Time course of iMOs in spleen during LACV infection in WT mice. (D) Illustration of transfer of Ccr2R/R fetal liver cells to irradiated MaFIA mice. Representative flow data for Ccr2+/+ (GFP) and Ccr2R/R (RFP) iMOs in blood of mock-infected (E) and LACV-infected (F) chimeric mice at 7 dpi. (G and H) IHC image of CCR2+ (GFP) and CCR2 (RFP) iMOs in the brain with staining for CD31+ BVs (white; asterisk) and LACV-infected neurons (blue). Arrows indicate GFP+ cells in close proximity to virus-infected neurons (blue).

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Analysis of brain tissue from LACV-infected BM chimeric mice revealed GFP+ CCR2+ iMOs and RFP+ CCR2 iMOs in the CNS (Fig. 3G, 3H). However, CCR2 iMOs remained primarily around blood vessels (BVs), whereas CCR2+ iMOs were observed away from BVs and near LACV-infected neurons (Fig. 3G, 3H, arrows). Additional IHC analysis of brains from LACV-infected Ccr2+/R mice also showed CCR2+ iMOs localized in areas of LACV infection throughout the brain parenchyma (Fig. 4A), whereas CCR2 iMOs in infected Ccr2R/R mice were found primarily around BVs (Fig. 4B) and did not migrate as far away from BVs (Fig. 4C). Costaining with CD31 (white staining) showed that CCR2 iMOs had crossed the brain endothelium into the perivascular space (Fig. 4B, inset). However, this lack of migration was not associated with any observable difference in infection or pathology in the brain. Because CCR2 binds CCL2, we examined whether CCL2-expressing cells were found in the brain during LACV infection. ISH for Ccl2 mRNA showed consistent staining around BVs, as well as GFAP+ cells, in the parenchyma, indicating that many of the Ccl2-producing cells are parenchymal astrocytes (Fig. 4D). These astrocytes may be responsible for recruiting CCR2+ iMOs into the brain parenchyma once they enter the CNS. In the absence of CCR2, there may not be signals sufficient to recruit iMOs to areas of infection within the brain. Thus, during LACV infection, CCR2 is not essential for recruitment of iMOs from the BM to the blood and CNS, but it does appear to be essential for migration of iMOs to the sites of infection within the brain. These studies suggest that the role of CCR2 in iMO recruitment and function is quite complex and varies among pathogens. In addition, it may vary among tissues. Because the exact mechanisms of CCR2-dependent iMO egress is not known, it is difficult to determine the mechanisms of LACV-induced CCR2-independent recruitment. However, studies with mice deficient in other chemokines, chemokine receptors (CXCL10, CCR5, CCR7), or initiators of innate immune responses (MyD88, MAVS) did not reveal altered iMO recruitment (Supplemental Fig. 2). This suggests that the CCR2-independent mechanism of iMO recruitment may be more complex than a simple compensatory mechanism that allows iMO egress.

FIGURE 4.

CCR2 promotes iMO dispersal away from perivascular areas into sites of LACV infection. IHC labeling of iMO (magenta) infiltration into LACV-infected (green) brains from Ccr2+/R (A) and Ccr2R/R (B) mice at 7 dpi. *Blood vessel. Inset in (B) shows iMOs in Ccr2R/R mice have crossed the CD31+ endothelium. DAPI = blue. Scale bar in (A) also pertains to (B). (C) Quantification of the distance from the center of CCR2+/R and CCR2R/R cell nuclei to the closest CD31+ BV (n = 2–5 mice per group with 12–19 20× fields analyzed) using Mann–Whitney analysis. ***p < 0.001. (D) ISH of LACV brain tissues at 7 dpi shows Ccl2 labeling (red) in GFAP+ astrocytes (brown, indicated by white arrows). Original magnification ×400.

FIGURE 4.

CCR2 promotes iMO dispersal away from perivascular areas into sites of LACV infection. IHC labeling of iMO (magenta) infiltration into LACV-infected (green) brains from Ccr2+/R (A) and Ccr2R/R (B) mice at 7 dpi. *Blood vessel. Inset in (B) shows iMOs in Ccr2R/R mice have crossed the CD31+ endothelium. DAPI = blue. Scale bar in (A) also pertains to (B). (C) Quantification of the distance from the center of CCR2+/R and CCR2R/R cell nuclei to the closest CD31+ BV (n = 2–5 mice per group with 12–19 20× fields analyzed) using Mann–Whitney analysis. ***p < 0.001. (D) ISH of LACV brain tissues at 7 dpi shows Ccl2 labeling (red) in GFAP+ astrocytes (brown, indicated by white arrows). Original magnification ×400.

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This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BM

bone marrow

BV

blood vessel

dpi

day postinfection

GFAP

glial fibrillary acidic protein

IHC

immunohistochemistry

iMO

inflammatory monocyte

ISH

in situ hybridization

JCV

Jamestown Canyon virus

LACV

La Crosse virus

MaFIA

Macrophage Fas-Induced Apoptosis

RFP

red fluorescent protein

TAHV

Tahyna virus

WNV

West Nile virus.

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The authors have no financial conflicts of interest.

Supplementary data