CD11b+, Ly6G+ Cells Produce Type I Interferon and Exhibit Tissue Protective Properties Following Peripheral Virus Infection

The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b+Ly6C+Ly6G- monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b+Ly6C+Ly6G+ cells. The phenotype of the CD11b+Ly6C+Ly6G+ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b+Ly6C+Ly6G+ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b+Ly6C+Ly6G+ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G+ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b+Ly6C+Ly6G+ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction

Type I interferon-dependent CCL4 is induced by a cGAS/STING pathway that bypasses viral inhibition and protects infected tissue, independent of viral burden.

Type I interferons (T1-IFN) are critical in the innate immune response, acting upon infected and uninfected cells to initiate an antiviral state by expressing genes that inhibit multiple stages of the lifecycle of many viruses. T1-IFN triggers the production of Interferon-Stimulated Genes (ISGs), activating an antiviral program that reduces virus replication. The importance of the T1-IFN response is highlighted by the evolution of viral evasion strategies to inhibit the production or action of T1-IFN in virus-infected cells. T1-IFN is produced via activation of pathogen sensors within infected cells, a process that is targeted by virus-encoded immunomodulatory molecules. This is probably best exemplified by the prototypic poxvirus, Vaccinia virus (VACV), which uses at least 6 different mechanisms to completely block the production of T1-IFN within infected cells in vitro. Yet, mice lacking aspects of T1-IFN signaling are often more susceptible to infection with many viruses, including VACV, than wild-type mice. How can these opposing findings be rationalized? The cytosolic DNA sensor cGAS has been implicated in immunity to VACV, but has yet to be linked to the production of T1-IFN in response to VACV infection. Indeed, there are two VACV-encoded proteins that effectively prevent cGAS-mediated activation of T1-IFN. We find that the majority of VACV-infected cells in vivo do not produce T1-IFN, but that a small subset of VACV-infected cells in vivo utilize cGAS to sense VACV and produce T1-IFN to protect infected mice. The protective effect of T1-IFN is not mediated via ISG-mediated control of virus replication. Rather, T1-IFN drives increased expression of CCL4, which recruits inflammatory monocytes that constrain the VACV lesion in a virus replication-independent manner by limiting spread within the tissue. Our findings have broad implications in our understanding of pathogen detection and viral evasion in vivo, and highlight a novel immune strategy to protect infected tissue

A systemic macrophage response is required to contain a peripheral poxvirus infection.

The goal of the innate immune system is to reduce pathogen spread prior to the initiation of an effective adaptive immune response. Following an infection at a peripheral site, virus typically drains through the lymph to the lymph node prior to entering the blood stream and being systemically disseminated. Therefore, there are three distinct spatial checkpoints at which intervention to prevent systemic spread of virus can occur, namely: 1) the site of infection, 2) the draining lymph node via filtration of lymph or 3) the systemic level via organs that filter the blood. We have previously shown that systemic depletion of phagocytic cells allows viral spread after dermal infection with Vaccinia virus (VACV), which infects naturally through the skin. Here we use multiple depletion methodologies to define both the spatial checkpoint and the identity of the cells that prevent systemic spread of VACV. Subcapsular sinus macrophages of the draining lymph node have been implicated as critical effectors in clearance of lymph borne viruses following peripheral infection. We find that monocyte populations recruited to the site of VACV infection play a critical role in control of local pathogenesis and tissue damage, but do not prevent dissemination of virus. Following infection with virulent VACV, the subcapsular sinus macrophages within the draining lymph node become infected, but are not exclusively required to prevent systemic spread. Rather, small doses of VACV enter the bloodstream and the function of systemic macrophages, but not dendritic cells, is required to prevent further spread. The results illustrate that a systemic innate response to a peripheral virus infection may be required to prevent widespread infection and pathology following infection with virulent viruses, such as poxviruses

Depletion methodologies during VACV infection.

<p>Depletion methodologies during VACV infection.</p

Splenic MZ macrophages are only depleted by conditions that allow systemic spread of VACV.

<p>Naïve LysMcre:iDTR mice (n = 8–12 from >3 experiments) were given a single injection of CLL i.v., DT i.p., or vehicle i.v.. At 1 day post-depletion, spleens <b>(A-F)</b> were isolated and cells extracted. Flow cytometry was used to count the total number of (<b>A</b>) macrophage/monocytes (F4/80⁺ CD11c^- CD11b⁺ Ly6G^-), (<b>B</b>) inflammatory monocytes (F4/80^- CD11c^- CD11b⁺ Ly6C⁺⁺ Ly6G^-), (<b>C</b>) neutrophils (F4/80^- CD11c^- CD11b⁺ Ly6C⁺ Ly6G⁺), (<b>D</b>) CD8⁺ DC (CD11c⁺ F4/80^- CD8⁺ CD11b^-), (<b>E</b>) DC (CD11c⁺ F4/80^-), and (<b>F</b>) CD11b⁺ DC (CD11c⁺ F4/80^- CD11b⁺ CD8^-). (<b>A</b>-<b>F</b>) In order to compile data across many experiments data are expressed as % of the mean number of cells in untreated mice. Results include all data from a minimum of 3 independent experiments (n = 8–14). (<b>G</b>) Naïve LysMcre:iDTR mice (n = 3–4) were given a single injection of CLL i.v. or i.d., DT i.p., or vehicle i.v. or i.d.. At 1 day post-depletion, LN were isolated and flash-frozen in OCT compound. Then 10–12 micron sections were fixed using acetone and stained with antibodies to CD169 (red) and SIGNR1 (green). Results are representative of those from 3 independent experiments (n = 8).</p

VACV infects splenic metallophilic MZ and MZ macrophages.

<p><b>(A)</b> C57BL/6 mice were infected i.v. with 100–100,000 pfu VACV and ovaries were harvested and titered at 5 days post-infection. <b>(B)</b> C57BL/6 mice were infected i.v. with 10,000 pfu VACV-GFP. Spleens were harvested at 6 hours post infection and flash-frozen in OCT compound. Then 10–12μm sections were fixed and stained with antibodies to CD169 (purple) and SIGNR1 (red). <b>(C)</b> Quantification of fluorescent microscopy of infected cell populations in the spleen at 6hr post infection with VACV-GFP. Results are representative of those from 3 independent experiments (n = 6).</p

Depletion of local myeloid cell populations increases local pathogenesis and tissue damage.

<p>Mice on the C57BL/6 background were infected intradermally with 10,000 pfu VACV in each ear pinna. <b>(A-C)</b> MaFIA mice (n > 8) were injected with CLL i.v. as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006435#ppat.1006435.g001" target="_blank">Fig 1A</a> or AP20187 i.p. on Day 0 pre-infection and Days 1, 3 and 4 post-infection. At 5 dpi, ear pinnae were isolated and cells extracted. Flow cytometry was used to count the number of CD11b⁺ Ly6C⁺⁺ Ly6G^- inflammatory macrophages, CD11b⁺ Ly6C⁺ Ly6G⁺ tissue-protective myeloid cells, and total CD11b⁺ myeloid cells. (<b>D-F)</b> LysMcre:iDTR mice (n = 4) were injected i.p. with DT (40 ng/g) or vehicle on the day of infection. At 5 dpi, ear pinnae were isolated and cells extracted. Flow cytometry was used to count the number of CD11b⁺ Ly6C⁺⁺ Ly6G^- inflammatory macrophages, CD11b⁺ Ly6C⁺ Ly6G⁺ tissue-protective myeloid cells, and total CD11b⁺ myeloid cells. (<b>A</b>-<b>F</b>) In order to compile data across many experiments, data are expressed as % of the mean number of cells in untreated mice. Results include all data from a minimum of 3 independent experiments (n = 8–14). <b>(G-J)</b> Mice were treated as follows and VACV lesion size measured daily. <b>(G)</b> MaFIA mice (n = 5) were injected with AP20187 or vehicle as described in Materials and Methods. <b>(H)</b> LysMcre:iDTR mice (n = 5) were injected with DT (40 ng/g) i.p. or vehicle on Day 0 pre-infection and Days 2, 5, 8, 11 and 14 post-infection. <b>(I)</b> C57BL/6 mice (n = 5) were injected with CLL or vehicle i.v. on Day 0 pre-infection and Days 1, 3, 4, 11 and 18 post-infection. <b>(J)</b> CCR2^-/- or C57BL/6 mice were infected with VACV as above. All VACV pathogenesis data is representative of 3 independent experiments. <b>(K)</b> At 5 dpi, ear pinnae were isolated and cells extracted from C57BL/6 or CCR2-/- mice. Flow cytometry was used to count the number of CD11b⁺ Ly6C⁺⁺ Ly6G^- inflammatory macrophages and CD11b⁺ Ly6C⁺ Ly6G⁺ tissue-protective myeloid cells. (<b>L</b>) C57BL/6 (treated or untreated with CLL), MaFIA or LysMcre:iDTR mice were infected as above. Ears were harvested 5 days post-infection and virus was quantified using a standard plaque assay.</p

Systemic macrophages, but not DC, are crucial for blocking VACV dissemination during infection that bypasses the D-LN.

<p>(<b>A–C</b>) Mice were treated as follows and at 5 days post-infection, ovaries were harvested and used in a plaque assay for VACV titer. (<b>A</b>) Naïve CD11ccre:iDTR mice (n = 8–10) were given a single injection of CLL i.v., DT (20 ng/g) i.p., or vehicle i.v. 24 hours pre-infection. <b>(B)</b> C57BL/6 or Batf3^-/- mice were infected i.v. with 1000 pfu VACV. As a positive control, half the C57BL/6 mice were pre-depleted with CLL i.v. (n = 6–10) <b>(C)</b> C57BL/6 mice were infected with 1000, 10,000 or 100,000 pfu VACV i.v. or i.d.. 24 hours pre-infection, mice were injected with 250 μl CLL or HBSS i.v.. (<b>D</b>) Mice were pre-depleted with CLL i.v. and infected i.v. with 1000 pfu VACV, then spleen or ovaries were harvested at 0, 2 or 5 days post infection and a plaque assay used to determine VACV titer.</p

Depletion of local myeloid cell populations does not allow systemic spread of VACV, but does affect survival following ECTV infection.

<p>Mice (n ≥ 5) on the C57BL/6 background were infected intradermally with 10,000 pfu VACV in each ear pinna. (A) LysMcre:iDTR mice were injected with CLL i.v., DT (40 ng/g) i.p., or vehicle i.p., on day 0 pre-infection and days 1, 3 and 4 post-infection. At 5 or 7 dpi, ovaries were harvested and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–14). (B) MaFIA mice were injected with CLL i.v. on Day 0 pre-infection and Days 1, 3 and 4 post-infection, or AP20187 as described in Materials and Methods. At 5 or 7 dpi, ovaries were harvested and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–14). (C) Ovaries were harvested from wild-type, CCR2^-/-, or CX3CR1^-/- mice 3, 5, 7 or 9 dpi and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–12). (D) Wild-type C57BL/6 mice were injected with CLL i.v. on Day 0 pre-infection, or the anti-Ly6G antibody 1A8 (50 μg/g) on Days -4, -2 and 0 pre-infection, or the anti-Thy1 antibody T24 (30μg/g) on day -1 pre-infection and 3 dpi. At 5 dpi, ovaries were harvested and used in a plaque assay for VACV titer. Results include data from 3 independent experiments (n = 9). (E-G) Mice were infected in the footpad with 3,000 pfu ECTV, and survival was monitored for 2 weeks post-infection. (E) LysMcre:iDTR or wild-type mice were injected i.p. with 40 ng/g DT or vehicle on days -3, -2, and -1 pre-infection and days 2, 5, 8 and 11 post-infection. (F) MaFIA mice or wild-type mice were injected i.p. with 10 μg/g AP20187 or vehicle as described in Materials and Methods. (G) C57BL/6 or CCR2^-/- mice were left undepleted and survival was monitored. All ECTV graphs represent pooled data from 2 to 4 experiments (n = 10–20).</p