Nitric oxide and TNFα are critical regulators of reversible lymph node vascular remodeling and adaptive immune response

Abstract

Lymph node (LN) vascular growth, at the level of the main arteriole, was recently characterized for the first time during infection. Arteriole diameter was shown to increase for at least seven days and to occur via a CD4(+) T cell dependent mechanism, with vascular expansion playing a critical role in regulating induction of adaptive immune response. Here, using intravital microscopy of the inguinal LN during herpes simplex type II (HSV-2) infection, the data provides the first studies that demonstrate arteriole expansion during infection is a reversible vascular event that occurs via eutrophic outward remodeling. Furthermore, using genetic ablation models, and pharmacological blockade, we reveal arteriole remodeling and LN hypertrophy to be dependent upon both endothelial nitric oxide synthase (eNOS) and TNFα expression. Additionally, we reveal transient changes in nitric oxide (NO) levels to be a notable feature of response to viral infection and LN vascular remodeling and provide evidence that mast cells are the critical source of TNFα required to drive arteriole remodeling. Overall, this study is the first to fully characterize LN arteriole vascular changes throughout the course of infection. It effectively reveals a novel role for NO and TNFα in LN cellularity and changes in LN vascularity, which represent key advances in understanding LN vascular physiology and adaptive immune response.

Figure 1. Resting arteriole diameter at days zero, five, seven, nine, and thirty-five (5 weeks) after i.vag. infection with TK^-HSV-2.

Resting vessel diameters were recorded after at least sixty minutes of equilibration with superfused physiological saline solution where vessel diameter, in which arterioles had consistent diameters for at least the last thirty minutes of equilibration (A). Maximal arteriole diameter (+SNP) at days zero, five, seven, nine, and thirty-five (5 weeks) Data represents mean diameter (A,B) ± SEM wherein n = 11 5, 5, 4, and 4 animals for days 0, 5, 7, 9, and 35 respectively (B). Percent vessel constriction with respect to resting vessel diameter of LN arteriole of wild-type at day zero, one, three, five, seven, nine, and thirty-five p.i. in response to PE applied in fold increases from 10⁻⁹ to 10⁻⁵M. Data represents percent constriction (A,C) ± SEM were n = 6 for day zero and n = 5for days one, three, five, seven, nine, and thirty-five (C). Representative images of maximal diameter arterioles following infection at days zero and seven after infection In each image the arteriole is indicated by green arrows (D). Representative images of isolated LN arteriole cross-sections H&E stained (E). *p<0.05, **p<0.01, and ***p<0.001.

Figure 2. Resting and maximal (+Nifedapine) arteriole diameter at days zero and seven after i.vag. infection in eNOS^-/- mice with TK^-HSV-2 (A).

Representative images of resting and maximal diameter eNOS^-/- arterioles at days zero and seven after infection where arterioles are indicated by green arrows (B). Resting and maximal (+SNP) arteriole diameter at days zero and seven after i.vag. infection with Tk^-HSV-2 in mice treated systemically with L-NAME (C). Resting and maximal vessel diameters (% of uninfected) of WT, eNOS^-/- and L-NAME treated mice. For eNOS^-/- n = 4 and 5 respectively for days zero and seven vessel diameters, and n = 4 for all PE data points (D). For L-NAME treatment, n = 4 for all data points. For WT n = 5 at day seven p.i.. In all cases, one arteriole was studied per animal. *p<0.05, **p<0.01, and ***p<0.001.

Figure 3. Representative images of isolated eNOS^-/- lymph nodes following infection at days zero, one, five, and seven after infection (A).

Lymph node weights of isolated eNOS^-/- lymph nodes following infection at days zero and seven (B).

Figure 4. Percentage of vessel constriction in response to perfusion of 100μM L-NAME solution over the vessel preparation.

Zero, one, three, and five days p.i. in C57 BL/6 mice (A). Zero, seven, nine, and thirty-five days p.i. in C57 BL/6 mice (B). Zero, three, and seven days p.i. in CD4^-/- mice (C). Data represents percent vessel constriction in diameter ± SEM. *p<0.05 **p<0.01, and ***p<0.001.

Figure 5. Resting arteriole diameter at days zero, three, and seven after i.vag. infection with TK^-HSV-2 in TNFα^-/- mice (A).

Resting vessel diameters were recorded after at least sixty minutes of equilibration with superfused physiological saline solution where vessel diameter, in which arterioles had consistent diameters for at least the last thirty minutes of equilibration. Maximal arteriole diameter (+SNP) at days zero, three, and seven (B). Data represents mean diameter (A,B) ± SEM wherein n =  5, 4, and 4 for days 0, 3, and 7, respectively. Representative images of resting diameter TNFα^-/- arterioles following infection at days zero and seven after infection (C). In each image the arteriole is indicated by green arrows. Relative comparison of resting and maximal diameter wild-type C57 BL/6 (WT) and TNFα^-/- mice at seven days post-infection (D). *p<0.05, **p<0.01, ***p<0.001, ns =  not significant.

Figure 6. Phenylephrine (PE) dose response (percent vessel constriction) from 10⁻⁹M to 10⁻⁵M at zero (n = 5), three (n = 4), and seven (n = 4) days p.i. in TNFα^-/- mice in comparison to WT C57 BL/6 mice (n = 5) (A).

Representative images of lymph node arterioles following at day zero p.i. prior to PE application and following superfusion of 10⁻⁹, 10⁻⁷, and 10⁻⁵M PE (B). In each image the arteriole is shown to the upper right of the LN venule and indicated by a green arrow (C). Representative images of H&E stained arteriole cross-sections in TNFα^-/- mice zero and seven days p.i. Percentage of vessel constriction in TNFα^-/- mice in response to perfusion of 100μM L-NAME at days zero (n = 5), three (n = 4), and seven (n = 4) following infection (D). Data represents mean vessel constriction (A, C) ± SEM where one arteriole was studied per animal. *p<0.05, **p<0.01, and ***p<0.001.

Figure 7. Resting and maximal (+30µM Nifedapine) arteriole diameter at zero (n = 4) and seven (n = 5) days after i.vag. infection in mast cell deficient mice (A).

Comparison of maximal arteriole diameter in wild-type (C57 BL/6) (n = 5) and mast cell deficient mice (n = 5) at day seven following infection (B). Representative images of lymph nodes from mast cell deficient mice at days zero and seven following infection (C). Data represents mean vessel diameter (A, B) ± SEM where one arteriole was studied per animal. *p<0.05, **p<0.01, and ***p<0.001.