Surfactant-associated protein A provides critical immunoprotection in neonatal mice

Abstract

The collectins surfactant-associated protein A (SP-A) and SP-D are components of innate immunity that are present before birth. Both proteins bind pathogens and assist in clearing infection. The significance of SP-A and SP-D as components of the neonatal immune system has not been investigated. To determine the role of SP-A and SP-D in neonatal immunity, wild-type, SP-A null, and SP-D null mice were bred in a bacterium-laden environment (corn dust bedding) or in a semisterile environment (cellulose fiber bedding). When reared in the corn dust bedding, SP-A null pups had significant mortality (P < 0.001) compared to both wild-type and SP-D null pups exposed to the same environment. The mortality of the SP-A null pups was associated with significant gastrointestinal tract pathology but little lung pathology. Moribund SP-A null newborn mice exhibited Bacillus sp. and Enterococcus sp. peritonitis. When the mother or newborn produced SP-A, newborn survival was significantly improved (P < 0.05) compared to the results when there was a complete absence of SP-A in both the mother and the pup. Significant sources of SP-A likely to protect a newborn include the neonatal lung and gastrointestinal tract but not the lactating mammary tissue of the mother. Furthermore, exogenous SP-A delivered by mouth to newborn SP-A null pups with SP-A null mothers improved newborn survival in the corn dust environment. Therefore, a lack of SP-D did not affect newborn survival, while SP-A produced by either the mother or the pup or oral exogenous SP-A significantly reduced newborn mortality associated with environmentally induced infection in SP-A null newborns.

FIG. 1.

Heterozygous breeding strategy. The offspring of C3HeB/FeJ SP-A^+/+ and C3H/Hen SP-A^−/− breeding pairs were used to generate SP-A^+/− breeders. In breeding pair cross A, the female was heterozygous for SP-A, and in cross B, the male was heterozygous for SP-A. These SP-A^+/− breeders were crossed with C3H/Hen SP-A null mice, thus separating the effects of maternal SP-A production from the effects of neonatal SP-A production on the offspring. The SP-A genotypes of mothers and offspring are indicated. The mother and pup SP-A phenotypes are indicated by a plus sign (able to produce SP-A) or a minus sign (not able to produce SP-A). For example, the phenotype of an SP-A^+/− mother with an SP-A^+/− pup resembled the wild-type phenotype, while an SP-A^+/− mother with an SP-A^−/− pup allowed us to study the influence of maternal SP-A on an SP-A null pup. An SP-A^−/− mother with an SP-A^+/− pup allow us to study the influence of endogenous SP-A from the pup in the absence of maternal SP-A, while an SP-A^−/− mother with an SP-A^−/− pup allow us to study the SP-A null phenotype. All animals used for this breeding strategy were exposed to batch B corn dust bedding. WT, wild type.

FIG. 2.

Representative photograph of thoracic and abdominal organs in an SP-A null mouse born and reared in corn dust bedding. The animal appeared to be critically ill at approximately 24 h of age. The abdominal and thoracic organs are labeled. Abnormal intestinal gas was observed throughout the intestines (asterisks). Bar = 5 mm.

FIG. 3.

Representative GI tracts from SP-A null and wild type (WT) mice born and reared in the corn dust environment. The mouse on the left died after 8.5 h of life, while the other animals were euthanized after 24 h of life. An asterisk indicates the stomach, and an arrow indicates the site of the proximal intestine leaving the stomach. The stomach of the wild-type mouse is full of white milk, while the stomachs of the SP-A null mice contain little milk and their proximal intestines are bile colored.

FIG. 4.

Structure of the proximal small bowel of 24-h-old wild-type and SP-A null mice born and reared in control and corn dust bedding environments. The proximal small bowel structures were similar in the two strains of mice born in control bedding. The only consistent structural alteration observed in the SP-A null mice that appeared to be critically ill in the corn dust bedding was dilation of the intestinal lumen (L) and a paucity of fecal matter. The images are representative of four to six mice per condition. Bar =100 μm.

FIG. 5.

Inflammatory changes in the GI tract of critically ill SP-A null mice exposed to corn dust bedding. Twenty-four-hour-old SP-A null mice born and reared in control or corn dust bedding were examined. Representative histological sections from stomachs (A, B, and E) and small intestines (C, D, and F) are shown. SP-A null mice born in corn dust bedding (B) frequently exhibited sloughing gastric epithelial cells with condensed nuclei (asterisk) compared to SP-A null mice born in control bedding (A). On average, we observed 0.80 ± 0.26 sloughing gastric cell/high-power field in the SP-A null mice, which is significantly more (P < 0.05) than we observed in the SP-A null mice exposed to control bedding (0.25 ± 0.13 cell/high-power field). An example of this is shown in panel E. Neutrophils were also observed within and marginating along the gastric blood vessels of critically ill SP-A null pups in corn dust bedding (arrows in panel B). Also, SP-A null mice reared in corn dust bedding frequently exhibited inflammatory changes in the proximal small bowel (D) compared to SP-A null mice reared in control bedding (C). Specifically, neutrophils were frequently observed within the blood vessels at the base of the villi and also outside blood vessel walls (black arrows in panel D). Additionally, collections of mononuclear cells were also observed only in the ill SP-A null newborns exposed to corn dust bedding (open arrow in panel D). These changes in the proximal small bowel are shown at a higher magnification in panel F. These inflammatory findings were not observed in any of the age-matched SP-A null mice born in control bedding. The images are representative of four to six mice per condition. Bar = 50 μm.

FIG. 6.

Kaplan-Meier survival curves for newborn mice born and reared in control bedding compared with curves for mice born and reared in corn dust bedding. The mothers' and pups' SP-A genotypes, the bedding, the number of mice per group, and the percent survival are indicated. (A) Survival of wild-type and SP-A null pups in control bedding. The survival data were not different for the wild-type and SP-A null pups. (B) Survival of wild-type and SP-A heterozygous pups (offspring of SP-A heterozygous dames) born and raised in corn dust bedding (batch B). The data demonstrate there was no difference in survival between heterozygous pups of heterozygous mothers and wild-type pups of wild-type mothers. (C) Survival of SP-A heterozygous and SP-A null offspring generated from the SP-A heterozygous breeding pairs (Fig. 1) born and reared in corn dust bedding (batch B) and survival curves from panels A and B. In panel C, three major outcome groups, indicated by brackets 1 to 3, were identified. These distinct groups are distinguished by the absence of exposure to corn dust (bracket 1), the presence of SP-A in the mother, the pup, or both plus exposure to corn dust bedding (bracket 2), and the complete absence of SP-A in the mother and the pup plus exposure to corn dust bedding (bracket 3). Wild-type pup survival was significantly reduced (P = 0.022) (one asterisk) when the pups were exposed to corn dust bedding compared to pups exposed to control bedding (71 and 89%, respectively). The survival rates of heterozygous pups or SP-A null pups with heterozygous mothers (bracket 2) were not statistically different from each other (P = 0.676). A pup's survival in corn dust bedding was significantly improved (P = 0.048) (two asterisks) if either the mother or the pup was heterozygous for SP-A compared to a complete absence of SP-A in both the pup and the mother.

FIG. 7.

Kaplan-Meier survival curves for SP-A null newborn mice with and without oral SP-A treatment. Newborn SP-A null mice born and reared in corn dust bedding were either given two doses of SP-A protein (5 μg/5 μl) (○) or the same volume of sterile PBS (▪) twice in the first 24 h of life. At 5 days of life, the mice treated with SP-A had a significantly better chance of survival (P = 0.027) than their littermates treated with diluent (PBS). At day 21 of life, this trend for improved survival in the mice that received SP-A compared to the SP-A null mice that did not receive SP-A continued (P = 0.034).