Figure 6. Contribution of Nod1 to Chemokine Production, Neutrophil Recruitment, and Phagocytosis.

(A) The effect of Nod1 on stimulation of macrophage inhibitory protein (MIP-2) in upper respiratory tract lavage fluid in response to Sp and co-colonization with Hi in Nod1^−/− or parental C57Bl/6 mice (WT). Box-and-whiskers plot indicates high and low values, median and interquartile ranges; n ≥ 13 mice from two independent experiments for each co-infected group. *p < 0.02 compared to PBS control.

(B) Effect of Nod1 on the neutrophil influx in response to bacteria following co-colonization. Immunofluorescence showing co-localization of Hi and Sp with neutrophils in the murine nasal spaces. Parental (i) or Nod1^−/− (ii) mice were co-colonized with Hi636 and Sp1121, and at 24 h post-inoculation, adjacent 5-μm frozen parasagittal tissue sections through the lateral nasal spaces of the same animal were stained with anti-capsular polysaccharide serum specific to type b Hi (αHib), type 23F Sp (α23F), or αLy6.G antibody to mouse neutrophils. Hematoxylin and eosin–stained parasagittal sections (H&E) show the neutrophil influx into the lumen between adjacent nasal turbinates. DAPI nuclear staining (blue). Magnification 200×.

(C) FACS analysis of the effect of Nod1 of the influx of activated neutrophils. Administration (i.p.) of casein with 10⁶ heat-inactivated Hi or FK-156 (1.0 μg/animal) was compared to casein alone for the effect on PECs expressing Ly6.G and the activation marker CD11b/CD18. Values represent the percent of total PECs expressing both markers and are means of three independent determinations ± SD.

(D) Killing of Sp by neutrophil-enriched PECs at a ratio of one bacterium to 25 effector cells obtained from C57Bl/6 (black bars) or congenic Nod1^−/− (grey bars) mice pretreated by i.p. administration of heat-inactivated whole Hi636 (HKHi). Where indicated, activated neutrophils were treated ex vivo with cytochalasin D, an inhibitor of actin polymerization, or DPI, an inhibitor of NADPH oxidase and the oxidative burst. Survival of intracellular Sp was based on bacterial counts following treatment with gentamicin sulfate. Values represent ≥ three independent determinations in duplicate ± SD.