The Internal Conduit System of the Swine Inverted Lymph Node

Abstract

Lymph nodes (LN) are the crossroad where naïve lymphocytes, peripheral antigens and antigen presenting cells contact together in order to mount an adaptive immune response. For this purpose, LN are highly organized convergent hubs of blood and lymphatic vessels that, in the case of B lymphocytes, lead to the B cell follicles. Herein take place the selection and maturation of B cell clones producing high affinity antibodies directed against various antigens. Whereas the knowledge on the murine and human LN distribution systems have reached an exquisite precision those last years, the organization of the antigens and cells circulation into the inverted porcine LN remains poorly described. Using up to date microscopy tools, we described the complex interconnections between afferent lymphatics and blood vessels, perifollicular macrophages, follicular B cells and efferent blood vessels. We observed that afferent lymphatic sinuses presented an asymmetric Lyve-1 expression similar to the one observed in murine LN, whereas specialized perifollicular sinuses connect the main afferent lymphatic sinus to the B cell follicles. Finally, whereas it was long though that mature B cells egress from the inverted LN in the T cell zone through HEV, our observations are in agreement with mature B cells accessing the efferent blood circulation in the efferent, subcapsular area. This understanding of the inverted porcine LN circuitry will allow a more accurate exploration of swine pathogens interactions with the immune cells inside the LN structures. Moreover, the mix between similarities and differences of porcine inverted LN circuitry with mouse and human normal LN shall enable to better apprehend the functions and malfunctions of normal LN from a new perspective.

Keywords: B lymphocytes; endothelial cell (EC); fluorescence imaging (FLI); follicle; lymph node (LN); second harmonic generation (SHG); swine (source: MeSH NLM); whole organ imaging.

Figure 1

Main features of swine inverted LN in HES and B cells/macrophage immunofluorescent staining on consecutive sections. Consecutive 10 µm thick sections of a tracheobronchial LN were stained using (A) HES or (B) anti-CD21 (red) and anti-CD169 (green) antibodies and DAPI nuclear staining. Dotted white lines delimited Trabeculae (Tb), dashed yellow lines delimited the efferent area (Eff Area). F: B cell Follicle. Aff area, Afferent area; Eff area, Efferent area. Whole LN pictures are individual images from 10x objective acquisitions, scale bar 1000 µm. Images are representative of 3 LN from different animals.

Figure 2

LN vessels and sinuses are delimited by endothelial cells expressing different levels of CD31, veCadherin and Lyve-1. Consecutive 10 µm thick sections from the same LN as in Figure 1 were stained with (A) anti-CD169, anti-veCadherin, anti-CD31 antibodies and DAPI or (B) anti-CD21, anti-Lyve-1, anti-CD31 antibodies and DAPI. Tb, Trabeculae; Eff Area, efferent area. F: B cell Follicle. Red arrows: T cell area veCadherin^pos vessels, pink arrows: Trabecular CD31^pos endothelial cells, yellow arrows: Trabecular Lyve-1^pos endothelial cells, double pink/yellow arrows: efferent area sinus endothelial cells, blue arrows: efferent area veCadherin^pos vessels, violet arrows: efferent sinus Lyve-1^pos/veCadherin^pos endothelial cells. The right pictures numbered 1 and 2 in A) and 3 and 4 in B) referred to enlargements of the framed regions 1 to 4 on the left large pictures. Whole LN pictures are individual images from 10x objective acquisitions, scale bar 500 µm. Images are representative of 3 LN from different animals.

Figure 3

Trabecular lymphatic sinus and B cell follicles relative positioning 10 µm thick sections of a tracheobronchial LN were stained using (A) anti-CD169, anti-Lyve-1, anti-CD31 antibodies and DAPI; (B) anti-CD11c, anti-Lyve-1, anti-MHC-II and DAPI; (C) anti-Pax5, anti-Lyve-1, anti-Bcl6 and DAPI, (D) anti-CD169, anti-Ki67, anti-CD21 and DAPI; (E) anti- CD21, anti-Ki67, anti-Pax5 and DAPI; (F) anti- Pax5, anti-Ki67, anti- Bcl6 and DAPI. F, follicle; Tb, Trabeculae; pfMθ, perifollicular macrophages. Light blue arrow: CD169^pos macrophages, light green arrows: CD11c^pos/MHC-II^high dendritic cells, light yellow arrows: centrocytes. (A, B), scale bar 50µm, (C–F) scale bar 100µm. Whole LN pictures are individual images from 10x objective acquisitions. Images are representative of 3 LN from different animals.

Figure 4

Trabecular sinus, perifollicular sinus and blood vessels interconnections in relation with the perifollicular macrophages. (A) Thick slice of tracheobronchial LN were cleared using iDISCO+ and (B–D) different area and magnifications of cleared LN slice stained with anti-CD169 and anti-CD31 antibodies. Imaged with multiphoton microscope A1RMP+ with an objective lens 25x and dual excitation at 960 nm and 1040nm. From 960 nm excitation, Second Harmonic generation (SHG) was acquired in blue channel in forward direction and fluorescence of Alexa Fluor 488 was acquired in green channel in backward direction. From 1040 nm excitation, fluorescence of Alexa Fluor 555 was acquired in red channel in backward direction. F, follicle; Tb, Trabeculae. Red arrows: T cell area blood endothelial cells, pink arrows: peri-trabecular lymphatic endothelial cells, dark blue arrows: perifollicular lymphatic endothelial cells, green arrows: perifollicular macrophages, orange arrows: lymphatic-blood capillaries connections. (A–C), scale bar 100µm.

Figure 5

Blood vessels are evenly distributed from B cell follicles neighborhood to the inside of the efferent area. Thick sections of tracheobronchial LN have been cleared with iDISCO+ and stained for CD169 (green) and CD31 (red). (A) wide view of CD169 (green) and CD31 (red) staining in order to delimit trabeculae (doted white lines) and efferent area (dashed yellow lines). F, Follicle; Tb, Trabeculae; TZ, T cell zone; Eff Area, efferent area. (B) Close up the white rectangle from (A), with depicture of CD31 (red) staining only. Images were acquired with confocal microscope LSM780. (A), Scale bar 500µm. (B), scale bar 100µm Images are representative of 2 LN from one animal.

Figure 6

HEV are present in T cell areas, whereas cells presenting mature B cells features are present in the efferent area. 10 µm thick sections of a tracheobronchial LN have been stained for (A) Wide view of CD31 (green), ERTR7 (red) and DAPI (blue) staining, (B) Close up from the white rectangle from (A). Eff Area, efferent area; TZ, T cell zone; F, follicle; Tb, Trabeculae. (C) Pax5 expression on CD21 and CD79α-positive B cells [see Supplementary Figure 1(A) ], or (D) Blimp-1 expression on CD21 and IgM positive B cells [see Supplementary Figure 1(B) ]. The fluorescence of Pax5 and Blimp-1 were measured using Zen Blue software in different area of the LN: T cell area (TZ), the B cell follicles (Follicle), and the efferent area (effArea). Each symbol represents fluorescence measured on one cell. Since the data were non normally distributed (normality tested using Shapiro-Wilk test) the non-parametric Mann-Whitney test has been chosen. Images are representative of 2 LN from two animals. (A) Scale bar 200 µm, (B) scale bar 10 µm. ** p<0.01, **** p<0.0001.

Figure 7

Schematic depicting of the lymphatic and blood flow according to immune cells compartments. Aff Lymphatic, Afferent lymphatic; Tb, Trabeculae; F, follicle; TZ, T cell zone; Eff Area, efferent area; Eff Sinus, efferent sinus; Eff Lymphatic, Efferent lymphatic; HEV, High endothelial venule; LEC, Lymphatic endothelial cells; pf Lymphatic, perifollicular lymphatic; Free Ag, Free antigen; DC-Ag, Antigen transported by DC; pfMθ, perifollicular macrophage.