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. 2022 Mar 23;9:849990. doi: 10.3389/fmed.2022.849990

Figure 6.

Figure 6

Comparison of γδT and dry eye signs in bone marrow chimeras. (A) Conjunctival goblet cell number in C57BL/6 mice exposed to desiccating stress (DS) for 5 days (DS5) with or without systemic treatment with anti-IL-17 neutralizing antibody or isotype control as described in the methods. (B) Representative flow cytometry plots of the donor (CD45.2+) and the recipient (CD45.1+) bone-marrow derived cells (left) and γδ TCR high and low CD3+ T cells in the conjunctivas of Pepc/BoyJ recipient (host) chimeric mice (age 8–10 weeks) reconstituted with B6 or Pinkie bone marrow after 5 days of desiccating stress. The method of chimera creation is provided in Supplementary Figure 3. (C) Top left: bar graph shows the percentage of CD45.2+CD3+γδTCR+ in the recipient conjunctiva (n = 11/group). Bottom left: histogram of percentage of IL-17+ cells from CD45.2+CD3+γδTCR+ gate in representative sample. Right: mean +/– SD percentage (top) and mean fluorescent intensity (bottom) of IL-17A+CD3+γδ TCR+ cells in the conjunctiva of chimeras (n = 11/group). (D) Confocal microscopy of whole-mount conjunctivas or cryosections obtained from B6 and Pinkie bone marrow chimeras created as shown in Supplementary Figure 3 with or without systemic treatment with anti-IL-17 neutralizing antibody or isotype control as described in the methods and exposed to DS for 5 days stained with an antibody specific for MMP-9 (top), evaluated for in situ gelatinase (zymography) activity in cryosections (middle), or stained with polyclonal antibody to cornified envelope precursor SPRR2 (bottom) (n = 3 per group). Bar graphs to the right show mean ± SD fluorescent intensity of the fluorochrome/fluorescent gelatin measured by Nikon Elements software (n = 3/group). (E) Conjunctival goblet cell number in Pinkie donor bone marrow chimeric mice exposed to desiccating stress (DS) for 5 days (DS5) with or without systemic treatment with anti-IL-17 neutralizing antibody or isotype control as described in the methods. Representative photomicrographs of periodic acid-stained sections for each treatment group (left) and graph of mean ± SD of goblet cells/mm (n = 5). Some goblet cells in the control group appear entrapped in the epithelium as previously reported (24).