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. 2024 Sep 17:15:1455238.
doi: 10.3389/fimmu.2024.1455238. eCollection 2024.

Podoplanin expressing macrophages and their involvement in tertiary lymphoid structures in mouse models of Sjögren's disease

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Podoplanin expressing macrophages and their involvement in tertiary lymphoid structures in mouse models of Sjögren's disease

Aud-Malin Karlsson Hovd et al. Front Immunol. .

Abstract

Tertiary lymphoid structures (TLSs) are formed in tissues targeted by chronic inflammation processes, such as infection and autoimmunity. In Sjögren's disease, the organization of immune cells into TLS is an important part of disease progression. Here, we investigated the dynamics of tissue resident macrophages in the induction and expansion of salivary gland TLS. We induced Sjögren's disease by cannulation of the submandibular glands of C57BL/6J mice with LucAdV5. In salivary gland tissues from these mice, we analyzed the different macrophage populations prior to cannulation on day 0 and on day 2, 5, 8, 16 and 23 post-infection using multicolored flow cytometry, mRNA gene analysis, and histological evaluation of tissue specific macrophages. The histological localization of macrophages in the LucAdV5 induced inflamed salivary glands was compared to salivary glands of NZBW/F1 lupus prone mice, a spontaneous mouse model of Sjögren's disease. The evaluation of the dynamics and changes in macrophage phenotype revealed that the podoplanin (PDPN) expressing CX3CR1+ macrophage population was increased in the salivary gland tissue during LucAdV5 induced inflammation. This PDPN+ CX3CR1+ macrophage population was, together with PDPN+CD206+ macrophages, observed to be localized in the parenchyma during the acute inflammation phase as well as surrounding the TLS structure in the later stages of inflammation. This suggests a dual role of tissue resident macrophages, contributing to both proinflammatory and anti-inflammatory processes, as well as their possible interactions with other immune cells within the inflamed tissue. These macrophages may be involved with lymphoid neogenesis, which is associated with disease severity and progression. In conclusion, our study substantiates the involvement of proinflammatory and regulatory macrophages in autoimmune pathology and underlines the possible multifaceted functions of macrophages in lymphoid cell organization.

Keywords: CD206; CX3CR1; Sjögren; autoimmunity; macrophages; mannose receptor; podoplanin; salivary gland.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
TLS development in LucAdV5-induced primary Sjögren’s disease model. Aggregation of T and B cells during LucAdV5-induced primary Sjögren’s disease was used to assess the development of TLSs in the salivary glands of C57BL/6J mouse model. Immunofluorescence (IF) labelling of CD45, CD3 and B220 was performed on salivary gland cryosections at six different time points during LucAdV5 infection. (A) Relative frequency of CD45+ cells in scanned tissue sections, based on the total number of cells detected in the tissue. (B) Relative frequency of B cells (CD45+B220+) and T cells (CD45+CD3+) based on the number of infiltrated CD45+ cells in the tissue section. (C) Relative frequencies of T cells and (D) B cells in three regions of interest. (E) Representative images of ROI 3; B and T cell aggregation for different time points during inflammation processes [CD45(white), CD3(green) and B220(red)]. Changes in gene expression of (F) Ccl21, (G) Cxcl13, (H) Foxp3, (I) Ltβ and (J) Ltβrare shown as fold changes. Data are presented as mean ± SEM. One-way ANOVA with the post-hoc analysis Dunnett’s multiple comparison test was used for panels (A, B, F–J), while two-way ANOVA statistical test with the post-hoc analysis and Tukey’s multiple comparisons tests were applied for panels (C, E) * equals a p-value <0.05, ** equals a p-value <0.01, *** equals a p-value <0.001, **** equals a p-value <0.0001. Non-statistically significant values are not shown. The statistics of the IF-stained tissue is based on cell segmentation and fluorescence classification performed with QuPath V-0.5.1.
Figure 2
Figure 2
Myeloid cell population response and dynamics in LucAdV5-induced inflammation with focus on the expression of CX3CR1. (A) Pie chart presentation of flow cytometric analyses showing the changes in myeloid cell populations studied in the submandibular salivary glands from before (day 0) and after (day 2, 5, 8, 16 and 23) infection of LucAdV5 in C57BL/6J mice. (B) Changes in the F4/80+ macrophage, CD11b+ DC2, CD11c+ DC1, F4/80- monocyte and neutrophil populations represented as the relative frequency of the myeloid cell population. (C) Relative frequencies of CX3CR1 positive cells in the populations of F4/80+ macrophages, CD11b+ cDC2, CD11c+ cDC1 and F4/80- monocytes. (D) Representative histogram of CX3CR1 expression in F4/80+ macrophage, cDC2, cDC1 and F4/80- monocyte populations. The expressions are shown as a percentage of the maximum count and the dashed line indicates the cut-off between positive and negative events. The data are presented as mean with +/- SEM. One-way ANOVA and post-hoc analysis Dunnett’s multiple comparison test was performed to test for differences between the populations prior to LucAdV5 infection (day 0) and the populations after LucAdV5 infection (day 2, 5, 8, 16 and 23). Statistically significant values are marked with “*”, where: * equals a p-value <0.05, ** equals a p-value <0.01, *** equals a p-value <0.001, **** equals a p-value <0.0001. Non-statistically significant values are not shown in the figures. The gating strategy of myeloid cell populations in the salivary glands is shown in Supplementary Figure 2 .
Figure 3
Figure 3
CX3CR1 expressing myeloid cells and their PDPN and CD206 phenotypes. Myeloid cell populations were gated into the tSNE plot analysis for the two panel setups of the flow cytometry experiments. (A) Myeloid cell clustering for panel 1, with emphasis on F4/80+ macrophages, F4/80- monocytes, cDC1 and cDC2. Heatmap visualization of CX3CR1 expression. (B) Heatmap visualization of PDPN expression during LucAdV5 infection. (C) Myeloid cell clustering for panel 2 and panel 1 with an emphasis on F4/80+ macrophages, F4/80- monocytes, cDC1 and cDC2. Heatmap visualization of CX3CR1 expression. (D) Heatmap visualization of CD206 expression during LucAdV5 infection. Relative frequencies of (E) CX3CR1+PDPN+ and (F) CX3CR1+CD206+ cells in the populations of F4/80+ macrophages, CD11b+ cDC2, CD11c+ cDC1 and F4/80- monocytes. Changes in gene expression of (G) Il1β, (H) Tnf, (I) Il18, (J) Ifny, (K) Mx1(Ifnα), (L) Il10, (M) Tgfβ, (N) Cd169, (O) CX3CR1, (P) Pdpn and (Q) Cd206 are shown as fold changes normalized to uninfected salivary gland tissue. The data are presented as mean with +/- SEM. One-way ANOVA and post-hoc analysis Dunnett’s multiple comparison test was performed to test for differences between the populations prior to LucAdV5 infection (day 0) and the populations after LucAdV5 infection (day 2, 5, 8, 16 and 23). Statistically significant values are marked with “*”, where: * equals a p-value <0.05, ** equals a p-value <0.01, *** equals a p-value <0.001, **** equals a p-value <0.0001. Non-statistically significant values are not shown in the figures.
Figure 4
Figure 4
Localization of F4/80+, PDPN+ and CD206+ cells in salivary gland tissue during LucAdv5 infection. Immunofluorescence labelling of F480, PDPN and CD206 expressing cells was performed on salivary gland cryosections at six different time points during LucAdV5 infection. (A) Relative frequencies of F4/80+PDPN+, F4/80+CD206+, F4/80+CD206+ PDPN+ and total F4/80+ cells in the scanned tissue section, based on the total number of cells detected in the tissue. (B) Representative images showing F4/80 (magenta), PDPN (green) and CD206 (yellow) labelling of the three regions of interest (ROIs) at different time points. The relative frequency of the F4/80 labelling profiles in (C) Region 1 “region with low inflammation”, (D) ROI 2 “Region with inflamed tissue”, (E) ROI 3 “Region with T and B cell aggregate”. Abbreviations salivary gland ducts (D), lymphatic vessel (LV) and tertiary lymphoid structure (TLS). Data presented as mean +/- SEM. Two way ANOVA and the post-hoc analysis Dunnett’s multiple comparison test were performed to test for differences between the populations prior to LucAdV5 infection (day 0) to the populations after LucAdV5 infection (day 2, 5, 8, 16 and 23. Statistically significant values are marked with “*”, where: * equals a p-value <0.05, ** equals a p-value <0.01, **** equals a p-value <0.0001. Non-statistically significant values were not included in the graphs. Images of the scanned IF-stained sections are shown in Supplementary Figure 1B . The statistics of IF-stained tissue were based on cell segmentation and fluorescence classification performed using QuPath V-0.5.1.
Figure 5
Figure 5
Organization of the F4/80+ macrophages in salivary gland tissue from NZBW-F1 mice with focus on their expression of PDPN and CD206. Immunofluorescence labelling of F480, PDPN, and CD206 expressing cells was performed on paraformaldehyde-fixed and paraffin-embedded salivary gland tissue from NZBW-F1 mice negative or positive for dsDNA autoantibodies, which are clinical markers of SLE. (A) Representative images of F4/80 (magenta), PDPN (green) and CD206 (yellow) labelling of the three different regions of interest (ROIs) in the salivary glands of NZBW-F1 mice. (B) Relative frequency of F4/80+PDPN+, F4/80+CD206+, F4/80+CD206+PDPN+ and total F4/80+ cells in whole scanned tissue section. Relative frequency of (C) F4/80+ cells, (D) F4/80+PDPN+, (E) 4/80+CD206+ and (F) F4/80+CD206+PDPN+ in the three different regions, ROI1 1 “region with low inflammation”, ROI 2 “Region with inflamed tissue”, and ROI 3 “Region with T and B cell aggregate”. (G) Representative image of salivary gland TLSs with (G2) and without (G1) overlay of the cell segmentation. Unclassified cells and cells marked only with CD206 or PDPN were excluded from the figure. White arrowheads points to F4/80+CD206+PDPN+ macrophages located within the TLS. Abbreviations salivary gland ducts (D), lymphatic vessel (LV) and tertiary lymphoid structure (TLS). Data presented as mean +/- SEM. Two-way ANOVA and post-hoc Tuckey’s multiple comparison tests were performed to test for differences. Statistically significant values are marked with “*”, where: * equals a p-value <0.05, ** equals a p-value <0.01, **** equals a p-value <0.0001. Non-statistically significant values are not included in the graphs. The statistics of IF-stained tissue were based on cell segmentation and fluorescence classification performed using QuPath V-0.5.1.

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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded in whole or in part by Northern Norway Regional Health Authority (HNF1427-18), UiT The Arctic University of Norway.

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