Macaque paneth cells express lymphoid chemokine CXCL13 and other antimicrobial peptides not previously described as expressed in intestinal crypts

Abstract

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), β-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.

Fig 1

Localization of CXCL13 mRNA and protein in macaque lymph node and spleen. CXCL13 protein was detected by IHC (brown signal) or immunofluorescent staining (LN, AIDS, top row) in lymph node (A) or spleen (B) at the indicated SIV-related disease state. Parallel controls performed with control antibody or cognate sense riboprobes are presented to the far right. Original magnifications, ×200.

Fig 2

Expression of CXCL13 mRNA and protein in intestinal crypts. IHC (brown signals) and ISH (black silver grain signals) were performed on macaque jejunum, revealing localization of CXCL13 producer cells in the intestinal crypts of Lieberkühn. Parallel controls performed with control antibody or sense riboprobes are shown in the bottom two images. Original magnifications, ×200.

Fig 3

Identification of Paneth cells as producers of CXCL13 and other antimicrobial peptides. (A) Phloxine-tartrazine staining was performed for Paneth cell granules in the crypts of Leiberkühn in macaque ileum. (B) IHC was performed to detect DEFA6, a marker for Paneth cells, in macaque ileum. The brown signal indicates the location of the bound antibodies. (C) ISH for DEFA6 mRNA (black silver grains) was combined with IHC for CXCL13 (brown signal) in macaque ileum. IHC for CXCL13 protein was combined with ISH for mRNAs encoding (D) DEFA1, (E) BDEF2, (F) RTD-1, or (G) Reg3γ in macaque ileum. The inset in panel D is a representative image following ISH with the corresponding sense control probe and IHC with an isotype control antibody. (H) Immunofluorescence staining and confocal microscopic detection of DEFA6 and CXCL13 was performed on macaque ileum.

Fig 4

Localization of antimicrobial peptide and CCL25 mRNAs in intestinal tissues. In situ hybridizations were performed to localize DEFA6 (A to C) and CCL25 (D to F) mRNAs in uninfected, acutely infected, and AIDS-developing macaque jejunum, ileum, and colon tissue sections. ISHs were also performed to detect BDEF2 (G), RTD-1 (H), and Reg3γ (Reg3g) (I) mRNAs in uninfected macaque jejunum. ISH results obtained with parallel hybridization with the cognate sense control riboprobes are shown in panels J to L. Original magnifications, ×100.

Fig 5

Measurement of DEFA6 mRNA levels in macaque ileum. Real-time RT-PCR (TaqMan) was used to measure DEFA6 mRNA levels in homogenized ileum, which was normalized to the endogenous control β-GUS, and then all values were calibrated to one uninfected sample. Shown are the individual values and the means for each group. Statistically significant differences were observed relative to the uninfected group (Mann-Whitney test): *, P < 0.05; **, P < 0.01.