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Link to original content: http://pubmed.ncbi.nlm.nih.gov/39188715/
Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation - PubMed Skip to main page content
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. 2024 Aug 12:15:1427509.
doi: 10.3389/fimmu.2024.1427509. eCollection 2024.

Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation

Andrea J Luker  1 , Abigail Wukitch  1 , Joseph M Kulinski  1 , Sundar Ganesan  2 , Juraj Kabat  2 , Justin Lack  3 , Pamela Frischmeyer-Guerrerio  1 , Dean D Metcalfe  1 , Ana Olivera  1
Affiliations

Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation

Andrea J Luker et al. Front Immunol. .

Abstract

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

Keywords: Sphingosine-1-phosphate receptor 4; germinal center reactions; inflammation; innate cells; lymph node hypertrophy; neutrophils.

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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
S1PR4-/- mice develop an attenuated GC response with diminished histological features. dLN from WT and S1PR4-/- mice were collected at various time-points after immunization with IFA/LPS/OVA in the hind footpad and analyzed for (A) total cellularity, (B) total number of GC B cells, and (C) total number of TFH cells. The dotted lines indicate approximated curves for the kinetics of GC responses in WT (red) and S1PR4-/- mice (blue). (D) Representative confocal images of WT and S1PR4-/- dLN of naïve (left; scale bar, 300 µm) or Day 9 post-immunized (right; scale bar, 400 µm) mice showing B cells (B220 in blue), T cells (CD4 in orange), and GC (PNA in green). Histological quantification of (E) total dLN area of tissue section from naïve and immunized mice, (F) number of GC regions per LN section, (G) individual GC area measurements, and (H) cumulative area of all GC per LN section. Region of Interest studies were performed within masked GC regions and analyzed for the percent and total number of (I) CD4+ and (J) PNA+ cells. Red and blue circles indicate WT and S1PR4-/-, respectively. Values represent LN from one individual mouse (E, F, H) or multiple GC measurements within WT or S1PR4-/- LN (G, I, J) from a total of 7-14 sections. Data represent Mean ± SD; ns, not statistically significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 using unpaired t-tests (A-D) or multiple unpaired t-tests (A-C).
Figure 2
Figure 2
Transcriptome profiling of sorted GC B cells and TFH cells after immunization indicates reduced activation in S1PR4-/- mice. (A) RNA-Seq analysis of GC B cells (S1PR4-/- vs WT) isolated nine days after immunization with IFA/LPS/OVA. Analysis was performed using QIAGEN IPA. The top Canonical Pathways with highest p-values are shown on the left and the predicted Z-scores indicated on top of the bars. Blue bar indicates predicted inhibition (negative Z-score), and white bar indicates no prediction of activation/inhibition. Genes affected in the pathways are noted above the bar graph. On the right panel, Z-Scores of Upstream Regulators predicted to be activated (orange bars) or inhibited (blue bars) in S1PR4-/- compared to WT GC B cells with downstream effects indicated on right. (B) Principal Component Analysis (PCA) illustrating transcriptional differences between TFH cells isolated from S1PR4-/- and WT dLN six days post-immunization. (C) Heatmap of differentially expressed genes (FDR< 0.05 and fold change > 1.5) in TFH cells (S1PR4-/- vs WT) from a subset of genes that are differentially expressed in TFH cells compared to non-T FH CD4+ T cells in WT mice (FDR< 0.05 and fold change > 1.5). The top molecular functions related to these genes are indicated on the bottom and were obtained in an enrichment analysis (ShinyGO, http://bioinformatics.sdstate.edu/go/). (D) Top Network relationships and predicted functional effects determined by IPA using the genes shown in (C) Upregulated genes in the dataset (S1PR4-/- vs WT) are in red; downregulated in green. Predicted inhibited state of molecules are in blue and activated in orange. The degree of changes in expression or activation state is graded by the intensity of the colors. Lines indicate predicted relationships between nodes (blue: leads to inhibition; orange: leads to activation; yellow indicates inconsistent with state of downstream molecule).
Figure 3
Figure 3
S1PR4 is required for early neutrophil mobilization and subsequent dLN hypertrophy. (A) dLN from naïve or immunized mice were evaluated for total cellularity (left), total lymphocytes (middle), and total antigen-respondent (Ki67+) lymphocytes (right). Indicated dotted line in all panels represents 105 cells to highlight the change in y-axis scale. (B) Total number of circulating leukocytes (left), neutrophils (middle), and lymphocytes (right) quantified by flow cytometry. Data are reported as absolute numbers per 100 µL of whole blood. (C, D) Neutrophils mediate early hypercellularity in dLN after immunization. To deplete neutrophils, WT and S1PR4-/- mice were injected with 200 µg anti-Ly6G or isotype control antibody two days prior and at the time of immunization to deplete neutrophils (C). dLN were collected 24 hrs later and evaluated for total cellularity (D). Red and blue circles indicate WT and S1PR4-/-, respectively, with a textured bar indicating the neutralizing antibody treatment group. Data represent Mean ± SD of a representative or combined experiment(s) including 4-6 mice and repeated at least three times with similar results; ns, not statistically significant; *p<0.05; **p<0.01; *** p<0.001 using unpaired t-tests.
Figure 4
Figure 4
Impaired neutrophil tissue infiltration in S1PR4-/- underlies reduced inflammation and local vascular changes after immunization. (A) Hind paws were collected 6 hrs post-immunization then stained with H&E to visualize infiltration by immune cells. The top image is an image of a naïve hind paw indicating with square annotations the mid-planter and heel regions, respectively. Below are representative fields of mid-planter (left) or heel (right) of WT and S1PR4-/- mice, as indicated, at 5X and 80X (insets) magnification, showing early infiltrates. Scale bars indicate 50, 250, 500 µm or 5 mm. (B, C) Evaluation of inflammatory infiltrates collected 3 hrs post-immunization via subcutaneous paw flush and analyzed by flow cytometry. (B) Percentage (left) and total numbers (right) of lymphoid and myeloid populations from naïve and immunized WT and S1PR4-/- mice, as indicated. (C) Differential analysis of the lymphoid and myeloid populations. (D) Caliper measurements of experimental and contralateral paw thickness from WT and S1PR4-/- mice 3 hrs post-immunization. (E) Vascular permeability at the immunization site was determined via Miles assay. Mice were injected with Evan’s Blue dye 3 hrs post-immunization and tissue collected at 6 hrs. The dye was extracted and OD measurements were performed at 610 nm. On the right panel, mice were treated with neutrophil-depletion antibodies (striped bars) following the protocol shown in Figure 3C prior to Evan’s Blue dye injection as in the left panel, then analyzed using a Two-Way ANOVA with multiple comparisons. (F) Quantification of vessel diameter from paw sections from (A) The data points correspond to individual vessels scored in a total of 4-5 sections, each representing a different mouse. Red and blue circles indicate WT and S1PR4-/-, respectively. Data in (B-F) represent Mean ± SD of a representative or combined experiment(s) including 4-6 mice and repeated at least three times with similar results; ns, not statistically significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 using an unpaired t-test unless otherwise stated.
Figure 5
Figure 5
S1PR4-deficiency impairs recruitment of lymphocytes in a lymphocyte-extrinsic manner, late accumulation of dendritic cells, and vascular network development within the dLN. (A, B) Fluorescently-labeled WT and S1PR4-/- splenocytes (mixed 1:1) were adoptively transferred into both WT and S1PR4-/- hosts six days post-immunization. 24 hrs later, the number of transferred cells was quantified within the contralateral or draining LN. Pink and blue bar colors indicate WT or S1PR4-/- host, respectively. Donor WT splenocytes are indicated by red circles and S1PR4-/- by blue circles. Data was analyzed using a Two-Way ANOVA with multiple comparisons. (C) Quantification of dLN DCs at various time-points post-immunization. (D) Representative images of Day 9 dLN with staining for vascular endothelium (CD31, red), B cells (IgD, blue), and T cells (CD4, tan); scale bar, 400 µm. Quantification of (E) total identifiable CD31+ vessel segments; and (F) lumen diameter of largest 20 vessels as a measurement of vasodilation. (G) Representative images showing co-localization of vascular endothelium (CD31, red) and HEV regions (MECA79, green) in Day 9 dLN. (H) Quantification of the Mean Fluorescence Intensity (MFI) of the CD31 and MECA79 staining from a total of 7-8 sections. Red and blue circles indicate WT and S1PR4-/-, respectively. Data shown in B-C includes combined results from at least three independent experiments and error bars represent Mean ± SD; ns, not statistically significant; **p<0.01 using multiple comparisons or multiple unpaired t-tests. Values (E, F) represents LN from one individual mouse or multiple measurements within WT or S1PR4-/- LN (H). Error bars represent Mean ± SD (E, F) or Mean ± SEM (H); ns, not statistically significant; *p<0.05; ****p<0.0001 using multiple unpaired t-tests unless otherwise stated.
Figure 6
Figure 6
Graphical summary showing the proposed role for S1PR4 in bridging acute inflammation with the downstream formation of a robust GC reaction. (1) Following footpad immunization with IFA/LPS/OVA, neutrophils rapidly mobilize and infiltrate into the site of injection. (2) Increased neutrophils in the tissue initiate an innate response that causes edema and other changes in the local microenvironment. (3) The on-going acute inflammation conveys signals downstream that affect the local vasculature in order to increase circulation toward the dLN. (4) In response, the dLN vascular network begins to expand and mature its HEV portals to accommodate the influx of naïve lymphocytes, thus allowing further entry. (5) The accumulation of circulating lymphocytes results in dLN hypertrophy and allows for the formation of robust GC reactions. In S1PR4-/- mice, the amplification of this immune cascade is impaired, in part, due to failed neutrophil recruitment. Consequently, these mice exhibit reduced immune activity that ultimately results in attenuated GC formation. The graphic depicts the differing WT and S1PR4-/- phenotypes in red and blue, respectively.
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by the Division of Intramural Research of NIAID, NIH.

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