Ocular mucosal CD11b+ and CD103+ mouse dendritic cells under normal conditions and in allergic immune responses

Abstract

Steady state dendritic cells (DC) found in non-lymphoid tissue sites under normal physiologic conditions play a pivotal role in triggering T cell responses upon immune provocation. CD11b+ and CD103+ DC have received considerable attention in this regard. However, still unknown is whether such CD11b+ and CD103+ DC even exist in the ocular mucosa, and if so, what functions they have in shaping immune responses. We herein identified in the ocular mucosa of normal wild-type (WT) and Flt3-/- mice the presence of a CD11b+ DC (i.e., CD11c+ MHCII+ CD11b+ CD103- F4/80+ Sirp-a+). CD103+ DC (i.e. CD11c+ MHCII+ CD11b low CD103+ CD8a+ DEC205+ Langerin+) were also present in WT, but not in Flt3-/- mice. These CD103+ DC expressed high levels of Id2 and Flt3 mRNA; whereas CD11b+ DC expressed high Irf4, Csfr, and Cx3cr1 mRNA. Additionally, the functions of these DC differed in response to allergic immune provocation. This was assessed utilizing a previously validated model, which includes transferring specific populations of exogenous DC into the ocular mucosa of ovalbumin (OVA)/alum-primed mice. Interestingly, in such mice, topical OVA instillation following engraftment of exogenous CD11b+ DC led to dominant allergic T cell responses and clinical signs of ocular allergy relative to those engrafted with CD103+ DC. Thus, although CD11b+ and CD103+ DC are both present in the normal ocular mucosa, the CD11b+ DC subset plays a dominant role in a mouse model of ocular allergy.

Figure 1. DC subsets in the ocular mucosa are consistent phenotypically with CD103+ and CD11b+ DC.

(A) Gating scheme for analyses of conj DC subsets. Single cell preparations of harvested conj were analyzed for expression of the indicated markers. Events were gated on for viable (DAPI-), singlet (data not shown), CD11c+, I-A/E+, and autofluorescent- (empty FITC channel) cells, and then examined for CD11b+ and CD103+ DC subsets. (B) The presence of steady state CD103+, but not CD11b+, DC in the ocular mucosa is Flt-3 dependent. The above gating scheme was used to analyze the presence of CD11b+ vs. CD103+ DC from lung, liver and conj of WT and Flt-3 KO mice. (A-B) This experiment was derived from pooled lung (n = 3 mice), liver (n = 3 mice), and conj (n = 10). (C) DC subsets of the ocular mucosa share a uniform phenotype with steady state CD103+ and CD11b+ DC. The aforementioned gating scheme was used to analyze CD11b+ vs. CD103+ DC and the mean fluorescence intensities (MFI) of indicated markers were plotted. Statistical significance (*p<0.05) was calculated by comparing MFI of CD11b+ vs. CD103+ DC from pooled lung (n = 3 mice), liver (n = 3 mice), and conj (n = 10). (A–C) Data shown are representative of 3 independent experiments.

Figure 2. Transcriptional programs expressed by ocular mucosa DC subsets are consistent with steady state CD103+ and CD11b+ DC.

Lung, liver and conj from WT mice were prepared into single cell suspensions. Aforementioned gating scheme for FACS sorting was used to isolate CD11b+ vs. CD103+ DC. Indicated genes were assayed for by qRT-PCR assayed in the sorted DC subsets. Statistical significance (*p<0.05) via student's t test was derived from assessing pooled lung (n = 3 mice), liver (n = 3 mice), and conj (n = 25). Data shown are representative of 2 independent experiments.

Figure 3. Secondary allergic T cell responses are augmented as a result of conjunctival engraftment of purified BM-derived CD11b+ DC.

(A) MACS sorted BM derived CD103+ and CD11b+ DC. (B) Naïve host mice were adoptively transferred with purified T cells from OVA/alum primed mice. Hosts were subsequently engrafted with purified BM-derived CD103+ or CD11b+ DC (1.0×10ˆ5 cells), or sham engrafted with HBSS, prior to OVA ocular instillations. T cells from eye draining LN were harvested and stimulated in vitro with OVA for subsequent intracellular flow cytometry analyses of the indicated cytokines. This experiment is derived from pooled LN collected from an n = 4 mice per group. Data are representative of 2 independent experiments. (C) Supernatant from parallel cultures were pooled and assessed in technical triplicates (*p<0.05) via ELISA for IL-13 and IFN-g.

Figure 4. BM-derived CD11b+ DC engrafted into the conjunctiva leads to exacerbated ocular allergy clinical signs.

Naïve hosts were adoptively transferred with T cells from OVA/alum-primed mice and hosts were subsequently engrafted with purified BM derived CD103+ or CD11b+ DC (1.0×10ˆ5 cells). OVA challenges were administered via ocular instillations once a day, and masked clinical scoring was performed once a day at 20 min, 6 hr and 24 hr post challenge. Statistical significance (*p<0.05) was derived from assessing CD11b+ vs. CD103+ DC clinical scores in an n = 4 per group. Data are representative of 2 independent experiments.

Figure 5. Lung CD11b+ DC engrafted heterotopically into the conjunctiva leads to exacerbated ocular allergy clinical signs.

(A) Gating scheme for FACS sorting of lung CD11b+ vs. CD103+ DC. Single-cell suspensions of pooled lung (from n = 10 mice) gated for viability dye-, singlet, CD11c+, I-A/E+, and autofluorescent- cells, and CD11b+ vs. CD103+ were isolated. (B) Naïve host mice were adoptively transferred with T cells from OVA/alum primed mice and engrafted with purified lung CD103+ or CD11b+ DC (6.0×10ˆ4 cells). OVA challenges were administered via ocular instillations once a day, and masked clinical scoring was performed once a day at 20 min, 6 hr and 24 hr post challenge. Statistical significance (*p<0.05) was derived from assessing CD11b+ vs. CD103+ DC clinical scores in an n = 4 per group. Data are representative of 2 independent experiments.