Neutrophils Exert a Suppressive Effect on Th1 Responses to Intracellular Pathogen <em>Brucella abortus</em>

<div><p>Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, <em>Brucella abortus</em> is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular <em>Brucella</em> infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.</p> </div

Bacterial loads, spleen weights and rate of change in CFUs per spleen and CFUs per spleen weight over time.

<p>(A) Mice were infected i.p. with 10⁶ CFUs, at indicated times CFU/spleen and spleen weights were determined. Background levels of PBS injected mice in each period (dashed line) are depicted. (B) Rate of change in CFU/spleen (Δ CFU/spleen) and rate of change in CFU/spleen weight (Δ CFU/grams of spleen) were calculated over time using the following equations: Δ CFU/spleen = mean CFUs 8 or 15 days/CFUs 5 days ± SD; Δ CFU/grams of spleen = CFU/grams of spleen 8 or 15 days/5 days ± SD. Values of p<0.05 (*) and p<0.01 (**) in relation to WT values are indicated above the bars. Experiment was repeated three times with similar results.</p

Effect of corticosteroid treatment from 4 weeks of age on Lat^Y136F knock-in mice.

<p>Representative histopathological appearance of salivary glands and pancreas in 6-week-old corticosteroid-treated and saline-treated control mice. Seven mice were treated with corticosteroid in 3 independent examinations (2, 4 and 1 mouse respectively), and 7 mice were treated with saline in 2 independent examinations (3 and 4 mice respectively). Arrows indicate inflammatory lesions in the salivary glands (A), pancreas (B) and kidneys (C) of control mice, and in the salivary glands (D), pancreas (E) and kidneys (F) of corticosteroid-treated mice. (G) Inflammation scores of saline- or corticosteroid-treated mice. Mean scores with SEM of the number of 7 mice of each group analyzed are shown. (H) Fibrosis scores of saline- or corticosteroid-treated mice. Arrows indicated representative inflammatory lesions. Mean scores with SEM of 7 mice of each group analyzed are shown. Bars in each panel showed 400 μm.</p

Activation of T and B lymphocytes and recruitment of monocytes in WT and neutropenic mice (Genista and PMN-depleted) at 8 days post-infection.

<p>Leukocytes from lymph nodes, were analyzed at 8 days post-infection using (A) CD4⁺/CD44⁺, (B) CD8⁺/CD44⁺, (C) B220⁺/CD95⁺ and (D) CD11b⁺/Ly6C⁺ cell markers. Numbers in parenthesis indicate the percentages of non-infected mice. The percentages of cells found in each of the specified gates are indicated. Values of p<0.05 (*), p<0.01 (**) are indicated.</p

Leukocytes in lymph nodes from infected and non-infected WT, PMN-depleted and Genista mice were analyzed by flow cytometry at 8 and 15 days of infection using CD4+/CD44+, CD8+/CD44+, B220+/CD95+, and CD11b+/Ly6C+ cell markers.

<p>The percentages of cells found in each of the specified gates are indicated.</p

Fibrosis and obliterative phlebitis existed in Lat^Y136F knock-in mice.

<p>Azan staining revealed fibrosis in salivary glands at 20 weeks of age (A; magnification: 100×), pancreas at 16 weeks of age (B; magnification: 100×), and kidney at 20 weeks of age of Lat^Y136F knock-in mice (C; magnification: 40×). Azan staining of salivary glands of 20-week-old WT mice was shown as a control (D; magnification: 100x). Obliterative phlebitis was seen in pancreas (E; HE staining, magnification: 40×, F; EVG staining, magnification: 40×), and in lung (G; HE staining, magnification: 40×, H; EVG staining, magnification: 40×). Arrows in E-H indicated obliterative phlebitis. Bars in each panel showed 60 μm. Fibrosis scores of the salivary glands (I), pancreas (J), and kidney (K) in the LatY136F knock-in and WT mice. Fibrosis scores of the salivary glands (F), pancreas (G), and kidney (H) in LatY136F knock-in (circles) and WT mice (triangles) at different ages. *p<0.05, **p-value<0.01; Mann–Whitney U-test.</p

Levels of cytokines detected over time.

<p>The levels of INF-γ, IL-6, TNF-α and IL-10 were determined in the sera of WT, PMN-depleted and Genista mice i.p. infected with 10⁶ CFUs of <i>B. abortus</i> 2308 at 5, 8 and 15 days post-infection. Background levels of PBS injected mice (dashed line) are depicted in each graphic. Values of p<0.05 (*), p<0.01 (**) are indicated.</p

Activation of T and B lymphocytes and recruitment of monocytes in WT and Genista mice at 15 days post-infection.

<p>Leukocytes from lymph nodes were analyzed at 15 days post-infection using (A) CD4⁺/CD44⁺, (B) CD8⁺/CD44⁺, (C) B220⁺/CD95⁺ and (D) CD11b⁺/Ly6C⁺ cell markers. Numbers in parenthesis indicate the percentages of non-infected mice. The percentages of cells found in each of the specified gates are indicated. Values of p<0.05 (*), p<0.01 (**) are indicated.</p

Recruitment of monocytes/DCs (CD11b+/CD11c+) in the blood at 15 days post-infection.

<p>Leukocytes from blood were analyzed after 15 days of infection using CD11b⁺/CD11c⁺ cell markers. Numbers in parenthesis indicate the percentages of non-infected mice. The percentages of cells found in each of the specified gates are indicated. Values of p<0.05 (*), p<0.01 (**) are indicated.</p

Lymphoid depletion, macrophage infiltration and granuloma formation becomes more prominent in spleens of PMN-depleted and Genista than in WT <i>Brucella</i> infected mice.

<p>Spleens from infected (10⁶ CFUs) and PBS-treated mice were fixed and stained with hematoxylin and eosin stain. The absence of PMNs in Genista or PMN-depleted non-infected mice did not affect the normal distribution of white pulp (WP) and red pulp (RP) or the overall histological structure of the spleen.</p