Figure 1.

Desiccation stress recruits monocytes into the conjunctiva in a CCR2 dependent manner. The gating strategy used for these experiments is provided in Supplementary Figure 1A . (A) Single cell suspensions prepared from conjunctivae of C57BL/6 (B6) and Ccr2^-/- strains were stained with anti-CD45, CD11b, MHCII, CD64, Ly6c and MerTK. Squares within the representative plots show frequency of CD45⁺CD11b⁺MHCII^-CD64⁺Ly6C^high (classical monocytes) and CD45⁺CD11b⁺MerTK⁺ (tissue resident macrophages) cells (n = 6/group); (B) Experimental design, bone marrow cells from Pepc/BoyJ (2x10^6) were adoptively transfer via intra-orbital injection at 2 day time intervals and the recipient mice either remained in normal environmental conditions (NS) or were exposed to desiccating stress for 5 days (DS5) (n = 6/group); (C) relative fold change in CCL2 mRNA after desiccating stress for 1 day (DS1) (n = 6/group); (D) Adoptively transferred cells were tracked by gating on CD45.1⁺, squares within the representative plots showing frequency of classical monocytes (top) and CD45⁺CD11b⁺MHCII⁺ cells (bottom). The bar graphs on the right side indicate the percentages of these cells with each dot representing one animal (n = 6/group); (E) Ccr2^-/-/Ccr2^+/+ bone marrow chimera. Bone marrow ablation in Pepc/BoyJ mice was accomplished with ¹³⁷Cs irradiation with 1300 cGy, followed by intraorbitally injection of 2x10^6 bone marrow cells from Ccr2^-/-/CD45.2 (2x10^6) or wild type B6 (Ccr2^+/+/CD45.2) donors. Flow cytometry scatter plot shows the number of endogenous (CD45.1) or transplanted (CD45.2) immune cells in the conjunctiva two weeks post transfer; (F) Donor CD45.2⁺CD11b⁺ myeloid cells were measured in the conjunctiva of Pepc/BoyJ recipient mice after exposure to desiccating stress for 5 days (n = 6/group). (G) Donor CD45.2⁺CD11b⁺MHCII⁺ cells were measured in the conjunctiva of Pepc/BoyJ recipient mice after exposure to desiccating stress for 5 days (n = 6/group). The error bars indicate the standard error of mean (SEM), Student T-test was used for statistical comparison. ***p < 0.0001.