Corneas can be successfully single- or double-depleted of conventional dendritic cells and macrophages.

<p>Two days prior to HSV-1 infection (single injection), or every 2 days (multiple injections, started on -2 day p.i.), diphtheria toxin (DT), clodronate liposome (CL) or both, were injected subconjunctivally (s.c.) into CD11c-DTR transgenic or C57BL/6 WT mice in order to deplete corneal cDCs, MΦs or both; C57BL/6 WT mice injected with the mixture of DT and control liposomes (without clodronate) were used as sham-depleted controls. On days 0, 3, 6 p.i., corneas (n = 5/time point) were collected for immunostaining. <b>A:</b> Representative micrographs showing that CD11c+ cells (cDCs) were depleted, but the F4/80+ cells (MΦs) were present after s.c. DT injection. <b>B:</b> MΦs were depleted, but cDCs were present after s.c. CL injection. <b>C:</b> cDCs and MΦs were double-depleted after s.c. injection of DT and CL. <b>D, E:</b> After a single injection of DT on day -2 p.i., cDCs were almost completely depleted on day 0 p.i. However, on day 3 p.i., corneas repopulated with cDCs to 60% of normal corneal levels, and increased to more than 80% on day 6 p.i.; with repeated injections of DT every 2 days, cDCs remained depleted by more than 95% on days 0, 3, 6 p.i. (D). MΦs (E) or cDCs & MΦs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137123#pone.0137123.s001" target="_blank">S1C Fig</a>) were similarly depleted with repeated s.c. injections of CL or DT and CL every 2 days. Each experiment was replicated three times. Data are shown as mean ± SD. Scale bar: 100 μm.</p

Corneal conventional dendritic cell depletion results in delayed and decreased systemic viral transmission and subsequent increase in survival rate after corneal HSV-1 infection.

<p>After corneal HSV-1 infection, trigeminal ganglions (TGs) or draining lymph nodes (dLNs) (n = 6/time point) were collected and HSV-1 mRNA levels were measured. The survival rates of infected mice were monitored (n = 20). <b>A, D:</b> On day 1 p.i, HSV mRNA was detected in the TGs of sham-depleted or MΦ-depleted mice, but not in cDC-depleted (single or double-depleted with MΦ) mice. Viral mRNA was detected on day 3 p.i. in the TGs of mice with corneal cDC-depletion, but was significantly lower as compared to sham-depleted and MΦ-depleted mice. <b>B, E:</b> On day 4 p.i, viral mRNA was detected in dLNs of sham-depleted or MΦ-depleted mice, but not in mice with corneal cDC-depletion. However, on day 5 p.i., viral mRNA was detected in dLNs of mice with corneal cDC-depletion, but at significantly lower levels than in sham-depleted controls. <b>C, F:</b> HSV mRNA was also detected in the contralateral TGs with 2 days delay, and corneal cDC-depletion resulted in delayed and decreased virus levels. <b>G:</b> Survival rates of mice with corneal cDC-depletion or mice with corneal cDC/MΦ double-depletion were significantly increased as compared to sham-depleted mice, or MΦ-depleted mice. Each experiment, including the survival experiments, was replicated three times. *<i>p</i><0.05, ^<b>#</b><i>p</i><0.01 compared with sham-depleted corneas (ANOVA-D, E, F, two-sided; Fisher Exact Probability Test-G). Data are shown as mean ± SD.</p

Corneal conventional dendritic cells play a pivotal role in the defense against HSV-1 virus, by reducing local corneal damage and preserving vision, at the cost of mediating systemic viral transmission.

<p>HSV-infected corneas demonstrate local viral replication, increased inflammatory cell infiltration, and corneal nerve damage, resulting in keratitis and corneal scarring. Infection of corneal nerves by HSV-1 may directly or indirectly be mediated through cDCs, resulting in transmission of the virus to the systemic compartment, including TGs, dLNs and the CNS, resulting in latency, or death. However, depletion of corneal cDCs results in increased viral proliferation, corneal inflammatory cells infiltration, nerve damage, and increased keratitis severity, scarring and blindness. Further, there are decreased corneal nerves infection, resulting in delayed and decreased systemic viral transmission to TGs and dLNs, and subsequent decrease in mortality. Collectively, corneal cDCs reduce local corneal damages and preserve vision after HSV infection, at the cost of mediating systemic viral systemic transmission resulting in death.</p

Corneal nerve damage in primary acute herpes simplex keratitis is due to viral proliferation and not corneal inflammation.

<p><b>A, B:</b> Corneal nerve density was significantly decreased in PSGL1-KO mice as compared to WT mice after HSV-1 infection (n = 5/time point). <b>C, D:</b> The number of corneal inflammatory cells was significantly decreased in PSGL1-KO mice compared to WT mice after HSV infection (n = 5/time points). <b>E, F:</b> Corneal cDC density was significantly decreased in PSGL1-KO mice compared to WT mice after HSV infection. The increase of cDCs in PSGL-1 KO mice as compared to normal controls suggests potential differentiation from progenitor cells or proliferation (n = 5/time point). <b>G, H:</b> The number of corneal MΦs was significantly decreased in PSGL1-KO mice as compared to WT mice after HSV infection (n = 5/time points). <b>I</b>: Corneal viral titers were significantly increased in PSGL1-KO mice compared with WT mice after HSV-1 infection (n = 5/time point). Each experiment was replicated three times. *<i>p</i><0.05 compared with WT; ^<b>#</b><i>p</i><0.05 compared with normal (Student’s t-test, two-sided). Data are shown as mean ± SD. Scale bar: 100 μm.</p

Herpes simplex keratitis results in significant increased conventional dendritic cell and macrophage density, as well as increased maturation of dendritic cells.

<p>Corneal inoculation was performed with HSV-1, or virus culture medium as sham controls, on days 1, 2, 4, 6, 8 p.i., Corneas (n = 5/time point) were excised and stained with antibodies against CD11c, F4/80, MHC-II; normal corneas were used as controls. <b>A:</b> Representative micrographs of CD11c (conventional dendritic cells [cDCs]) and MHC-II (maturation marker) expressing cells in the HSV-1 infected corneas compared with sham-infected corneas. <b>B:</b> As early as day 1 p.i., cDC densities of central and peripheral corneas were significantly increased as compared to sham controls, and continuously increased on days 2, 4, 6, 8 p.i. <b>C:</b> cDC maturation (CD11c+MHC-II+/total CD11c+) after HSV-1 infection significantly increased vs. sham controls. <b>D:</b> Representative micrographs of F4/80 expressing cells (macrophages [MΦs]) in HSV-1 infected corneas compared with sham controls. <b>E:</b> MΦ density were significantly increased in central and peripheral corneas after HSV-1 infection as compared to sham controls on days 2, 4, 6, 8 p.i. <b>F, G:</b> The relative increase in density (density after HSV infection /density after sham infection) of cDC was significant higher than the relative increase in MΦ density in central corneas on days 1, 2, 4, 6, 8 p.i., while no significant differences were found in peripheral corneas. Each experiment was replicated three times. *<i>p</i><0.05, **<i>p</i><0.01 compared to sham controls (Student’s t-test, two-sided); data are shown as mean ± SD. Scale bar: 100 μm.</p

Upon depletion of corneal conventional dendritic cells, virus titers and the local corneal damages are significantly increased in primary herpes simplex keratitis.

<p>Infected corneas (n = 5/time point) were collected and virus titers, HSV mRNA levels measurement, or immunohistochemistry studies were performed. cDC and MΦ depletion were compared to sham-depleted infected mice after subconjunctival injections of DT and CL into CD11c-GFP-DTR or C57BL/6 WT mice, as well as to sham-infected mice without active viral inoculation (culture medium alone). Keratitis severity was graded (n = 5/time point). <b>A:</b> Representative corneal photos of primary acute herpes simplex keratitis severity of the mice in different groups are shown. <b>B:</b> Representative micrographs of viral invasion into the corneal stroma of the mice in different groups are shown. <b>C:</b> Representative micrographs of inflammatory cells (CD45⁺) infiltrating into the corneas of the mice in different groups are shown. <b>D:</b> Representative micrographs of nerve destructions of the mice in different groups are shown. <b>E:</b> The keratitis severity after HSV inoculation in cDC depleted (cDC(-)) mice was significantly increased as compared sham-depleted and MΦ-depleted (MΦ(-)) mice. The keratitis severity with cDC/MΦ double-depletion (cDC(-)&MΦ(-)) was further increased compared to cDC(-) mice. <b>F, G:</b> The viral titer levels (day 5 p.i.) of corneas and HSV-1 mRNA of the corneal stroma were significantly increased in cDC(-) corneas compared to sham controls or MΦ depletion. Double-depletion of cDCs and MΦs showed significantly HSV levels compared to cDC depletion alone. <b>H:</b> Increased inflammatory (CD45+) cells infiltrated corneas with cDC depletion (day 5 p.i.) compared to controls. cDC and MΦ double depletion resulted in increased CD45+ cells compared to cDC depletion alone. <b>I:</b> Corneal nerve density was significantly decreased in cDC depleted or cDC and MΦ double-depleted corneas as compared to sham controls or MΦ depleted corneas. Each experiment was replicated three times. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 compared with sham-depleted; ^<b>#</b><i>p</i><0.05 compared with cDC depletion (ANOVA with Bonferroni’s post-hoc test, two-sided). Data are shown as mean ± SD. Scale bar: 100 μm.</p

<i>V. cholerae</i> aggregates are present in the lumen of the lower SI, often associated with mucus.

<p>(A) Each panel shows lumenal aggregates of <i>V. cholerae</i> (VcRed and VcGreen) from the medial or distal SI. Colonies in the right panel reside on the surface of digesta. Scale bar = 100 µm. (B) Confocal microscopy images of tissue sections from the lower SI of infant mice inoculated with LB (control), VcGreen or the GFP-labeled cholera toxin–deficient mutant <i>ΔctxAB</i>-GFP. Sections were counterstained with wheat germ agglutinin (red) to visualize mucus and DAPI (blue) to stain nuclei. Scale bar = 25 µm.</p

Detection of fluorescently labeled <i>V. cholerae</i> in the intact infant mouse small intestine by intravital two-photon microscopy.

<p>(a) Schematic representation (left) of the surgical intravital imaging approach to visualize intestinal tissue by two-photo microscopy in live anesthetized mice following orogastric inoculation with <i>V. cholerae</i>. Transverse view of exteriorized intestinal loop (right), depicting objective focal path from the serosal side through the various tissue layers towards the intestinal villus. The resulting focal plane is a tangential section along the intestine as depicted in (b–d). (b) Sequential two-photon micrographs (XY plane) taken from a 3-dimensional Z-stack of the SI, revealing penetration depths for two-photon excitation of up to 150 µm below the serosa layer. Depths (Z axis) 0 to 50 µm below the serosa include crypt regions, and >100 µm encompass the villi region. Blue denotes collagen fibers, Magenta denotes blood vessels. Scale bar 50 µm. (c) Differential localization of <i>V. cholerae</i> within the crypt or villus regions in the proximal, medial, and distal segments of the SI. Top row panel displays representative intravital two-photon micrographs of the crypt region for the proximal SI segment, and of the villus region for the medial and distal SI segments, of non-infected mice. Bottom row panel displays representative intravital two-photon micrographs of the crypt region and villus regions for the proximal, medial, and distal regions of the SI following orogastric inoculation with VcGreen and VcRed, respectively. Blue denotes collagen fibers, Green denotes colonies of VcGreen, Red denotes colonies of VcRed. Scale bar 10 µm. (d) Visualization of VcGreen in explanted distal SI segments. Representative two-photon micrographs taken from time-lapsed movies (see Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004405#ppat.1004405.s008" target="_blank">Video S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004405#ppat.1004405.s009" target="_blank">Video S2</a>) show single VcGreen cells in the intravillus space and discrete microcolonies of VcGreen associated with the villus epithelium. White/Yellow denotes autofluorescence, Green denotes VcGreen. Scale bar 10 µm.</p

Distribution of fluorescently labeled <i>V. cholerae</i> in the infant mouse small intestine.

<p>(A) The small intestines of infant mice co-inoculated with VcRed and VcGreen were divided into three equal parts and the central 1 cm segments of the proximal (P), medial (M) and distal (D) parts were used for plating and microscopic analyses. (B) Numbers of CFUs recovered from homogenates of the proximal (P), medial (M) and distal (D) segments. Bars represent the geometric mean. *, p<0.05, and **, p<0.01, based on ANOVA with Tukey's multiple comparison test. (CEG) Confocal micrographs showing VcRed and VcGreen distribution in the proximal (C), medial (E) and distal (G) segments. Tissue sections were counterstained with phalloidin (blue) to visualize the surface of the epithelium. White arrowheads mark clonal microcolonies. Scale bar = 100 µm. (DFH) The distance separating microcolonies from the base of the villi in the proximal (D), medial (F) and distal (H) SI segments was measured using confocal microscopy in tissue cross sections from mice co-inoculated with VcRed and VcGreen. Data represent the mean ±SD from three mice. The number (n) of microcolonies analyzed is indicated to the left of each panel. Black triangles indicate the average length of villi in each region. (I) Average length of intestinal villi (±SD) in the proximal (P), medial (M) and distal (D) segments. ***, p<0.001, based on ANOVA with Bonferroni's multiple comparison test.</p

The intensity of WGA staining of the intestinal epithelium decreases along the length of the SI and can be reduced by N-acetyl-L-cysteine (NAC) treatment.

<p>(A) Confocal micrographs of longitudinal sections of the proximal, medial and distal SI from infant mice treated with PBS or NAC after 6 h. The sections were stained with WGA (red) and DAPI (blue). Scale bar = 200 µm. (B,C) PAS stained sections of Carnoy's fixed proximal, medial and distal SI segments from untreated infant mice (B) and of the proximal SI from infant mice treated with PBS or NAC after 6 h (C). Scale bar = 100 µm. Arrow points to mucus layer.</p