Fig 1: Production of CCL28 chemokines in the vaginal mucosa of HSV-2-infected SYMP and ASYMP B6 mice.(A) Experimental plan showing B6 mice (n = 20) were infected intra-vaginally (IVAG) with 2 × 105 pfu of HSV-2 (strain MS). The severity of genital herpes disease was scored for 14 days to segregate mice into SYMP or ASYMP groups, as described in the Material and Methods. On day 14 post-infection (dpi), SYMP and ASYMP mice and non-infected naïve mice (controls were euthanized and the vaginal mucosae were harvested and cell extracts were assayed by flow cytometry for frequencies of CD8+ T cells expressing CCR10, the receptor of CCL28 (i.e., CCR10+CD8+ T cells), and for CCL28 chemokine using IHC and ELISA. (B) Frequency of CCR10+CD8+ cells among total VM cells determined by flow cytometry in individual HSV-infected ASYMP (n = 4), SYMP (n = 4), and control non-infected (naïve) (n = 8) B6 mice. (C) The level of CCL28 chemokine quantified by ELISA (Abcam kit: ab210578) in the VM lysates of HSV-infected symptomatic (SYMP) B6 mice, HSV-infected asymptomatic B6 mice (ASYMP), and non-infected control B6 mice (Naïve). VM lysates from each mouse (n = 3) were pooled for this experiment. (D) Immunohistochemical staining of CCL28 (green) and DAPI (blue) in VM sections harvested on day 8 post-infection (dpi), from ASYMP, SYMP, and Naïve B6 mice. The lower panel shows a graph summarizing the fluorescence intensity (quantitated using Fiji) for CCL28 in the VM of mice. (E) Immunoblot of VM lysates from ASYMP, SYMP, and Naïve B8 mice (n = 3) probed using western blot for CCL28 (Abcam mAb clone ab23155) (top panel). The relative intensity of CCL28 normalized to b-actin is shown in the bottom panel. The results are representative of two independent experiments. The indicated P values are calculated using the unpaired t-test, comparing results obtained in SYMP vs. ASYMP and results obtained in ASYMP vs. Naïve mice.
Fig 2: Frequencies of central and effector memory CD44+CD8+ and CD44+CD4+ T cells in the vaginal mucosa of CCL28(-/-) knockout mice and B6 wild-type mice following intravaginal infection and re-infection with HSV-2.CCL28 KO mice (CCL28-/-) and WT B6 mice (n = 20) were IVAG infected with 5 × 103 pfu of HSV-2 strain 186 and then re-infected with 5 × 103 pfu of the same strain of HSV-2 on day 28 p.i. On day 10 post-re-infection, mice were euthanized and the frequencies of central memory CD44+CD62L+CD8+ TCM cells and CD44+CD62L+CD4+ TCM cells and of effector memory CD44+CD62L-CD8+ TEM cells and CD44+CD62L-CD4+ TEM cells were compared in the vaginal mucosa of CCL28 KO mice (CCL28-/-) and WT B6 mice using flow cytometry. (A) Representative data of the frequencies of total memory CD8+ T cells (top 2 panels) and central memory CD44+CD62L+CD8+ and effector memory CD44+CD62L-CD8+ T cells (middle 2 panels) in VM of CCL28 KO mice (CCL28-/-) and WT B6 mice re-infected with HSV-2. Average frequencies of total memory CD8+ T cells and central memory CD44+CD62L+CD8+TCM cells and effector memory CD44+CD62L-CD8+ TEM cells (bottom panel) in the VM of CCL28-/- and WT B6 mice are re-infected with HSV-2. (B) Representative data of the frequencies of total memory CD4+ T cells (top 2 panels) and central memory CD44+CD62L+CD4+ and effector memory CD44+CD62L-CD4+ T cells (middle 2 panels) in VM of CCL28 KO mice (CCL28-/-) and WT B6 mice re-infected with HSV-2. Average frequencies of total memory CD4+ T cells and central memory CD44+CD62L+CD4+TCM cells and effector memory CD44+CD62L-CD4+ TEM cells (bottom panel) in the VM of CCL28-/- and WT B6 mice are re-infected with HSV-2. The indicated P values were calculated using the unpaired t-test and compared results obtained from CCL28(-/-) (n = 3) and WT mice (n = 3) and the results are representative of two independent experiments.
Fig 3: Susceptibility of CCL28(-/-) knockout mice and B6 wild-type mice to genital herpes infection and disease following intravaginal infection and re-infection with HSV-2.(A) CCL28 KO mice (n = 12) and WT B6 mice (n = 12) were infected with IVAG with 5 × 103 pfu of HSV-2 (strain 186). CCL28 KO and WT B6 mice were scored every day for 8 to 9 days p. I for symptoms of genital herpes and severity of genital herpes scored, as described in Material and Methods. The disease was scored as 0- no disease, 2- swelling and redness of external vagina, 3- severe swelling and redness of vagina and surrounding tissue and hair loss in the genital area, 4- ulceration and hair loss in the genital and surrounding tissue. The vaginal swabs were collected on days 3, 5, and 7 p. I to determine virus titers. (B) Disease scoring in CCL28 KO mice (CCL28-/-) (n = 12) and WT B6 mice (WT) (n = 12) was determined for 9 days after primary infection with HSV-2 strain 186 (left panel). The maximal disease severity in CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) was determined 8 days after primary infection with HSV-2 strain 186(right panel). (C) Survival graph of in CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) determined for 14 days after primary infection with HSV-2. (D) The graph shows the virus titers detected in the vaginal swabs of CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) collected on 3-, 5-, 7-, and 10-days post-primary infection with HSV-2. (E) Representative pictures of genital disease in CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) taken on day 8 post-primary infection with HSV-2. (F) CCL28 KO mice (n = 3) and WT B6 mice (n = 3) were re-infected with IVAG with 5 × 103 pfu of HSV-2 (strain 186) on day 28 post-primary infection. Disease scoring in CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) was determined for 9 days after secondary re-infection with HSV-2 strain 186 (left panel). The maximal disease severity in CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) was determined 8 days after secondary re-infection with HSV-2 (right panel). (G) The graph shows the virus titers detected in the vaginal swabs of CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) collected 5-, 7-, and 10-day post-secondary infection with HSV-2. (H) Representative pictures of genital disease in CCL28 KO mice (CCL28-/-) and WT B6 mice (WT) taken on day 8 post-secondary infection with HSV-2. The results are representative of two independent experiments. The indicated P values were calculated using the unpaired t-test and compared results obtained from CCL28 KO mice (CCL28-/-) and WT B6 mice (WT).
Fig 4: Frequencies of CD8+ and CD4+ T cells expressing CCR10, the receptor of CCL28, in the vaginal mucosa of CCL28(-/-) knockout mice and B6 wild-type mice following intravaginal infection and re-infection with HSV-2.CCL28 KO mice (CCL28-/-) and WT B6 mice (n = 20) were IVAG infected with 5 × 103 pfu of HSV-2 strain 186 and then re-infected with 5 × 103 pfu of the same strain of HSV-2 on day 28 p.i. On day 10 post-final and secondary infection, mice were euthanized, and cell suspension from the vaginal mucosa (VM) and spleen was analyzed by flow cytometry for frequencies of CD8+ and CD4+ T cells expressing CCR10, the receptor of CCL28. (A) Representative and average frequencies of total CD8+ T cells (left panels) and total CD4+ T cells (right panels) in the VM of CCL28 KO mice (CCL28-/-) (n = 3) and WT B6 mice (n = 3) 10 days following re-infection with HSV-2. (B) Average frequencies of total CCR10+ T cells (top panels), CCR10+CD8+ T cells (middle panels), and CCR10+CD4+ T cells (bottom panels) detected in the VM (right panels) and spleen (left panels) of CCL28 KO mice (CCL28-/-) and WT B6 mice 10 days following re-infection with HSV-2. The results are representative of two independent experiments. The indicated P values were calculated using the unpaired t-test and compared results obtained from CCL28(-/-) and WT mice.
Fig 5: Frequencies of total B cells and memory B cells in the vaginal mucosa of CCL28(-/-) knockout mice and B6 wild-type mice following intravaginal infection and re-infection with HSV-2.CCL28 KO mice (CCL28-/-) and WT B6 mice (n = 20) were IVAG infected with 5 × 103 pfu of HSV-2 strain 186 and then re-infected with 5 × 103 pfu of the same strain of HSV-2 on day 28 p.i. On day 10 post-re-infection, mice were euthanized and the frequencies of total B220+B cells and memory B220+B cells, expressing the expressing CCR10, the receptor of CCL28, were determined for flow cytometry in the VM and spleen of CCL28 KO mice and WT B6 mice. (A) Representative (left 4 panels) and average (right 2 panels) frequencies of total B220+B cells (top 3 panels) and memory B cells (bottom 3 panels) in VM of CCL28 KO mice (n = 3) and WT B6 mice (n = 3), 10 days following re-infection with HSV-2. (B) Representative (left 4 panels) and average (right 2 panels) frequencies of total B cells expressing CCR10, the receptor of CCL28, (CCR10+B220+ B cells top 3 panels), and of memory B cells expressing CCR10 (CCR10+B220+CD27+ memory B cells, bottom 3 panels) were determined in the VM of CCL28 KO mice and WT B6 mice 10 days following re-infection with HSV-2. (C) The ELISPOT images show IgA ASC in the VM (top) and spleen (middle) of CCL28 KO mice and WT B6 mice 10 days following re-infection with HSV-2. Corresponding average SFU for IgA ASC in the VM and Spleen are shown in the 2 bottom panels. The results are representative of two independent experiments. P values were calculated using the unpaired t-test and compared with results obtained in CCL28 KO mice and WT B6 mice.
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