The Inherited Intestinal Microbiota from Myeloid-Specific ZIP8KO Mice Impairs Pulmonary Host Defense against Pneumococcal Pneumonia

Abstract

Intestinal dysbiosis increases susceptibility to infection through the alteration of metabolic profiles, which increases morbidity. Zinc (Zn) homeostasis in mammals is tightly regulated by 24 Zn transporters. ZIP8 is unique in that it is required by myeloid cells to maintain proper host defense against bacterial pneumonia. In addition, a frequently occurring ZIP8 defective variant (SLC39A8 rs13107325) is strongly associated with inflammation-based disorders and bacterial infection. In this study, we developed a novel model to study the effects of ZIP8-mediated intestinal dysbiosis on pulmonary host defense independent of the genetic effects. Cecal microbial communities from a myeloid-specific Zip8 knockout mouse model were transplanted into germ-free mice. Conventionalized ZIP8KO-microbiota mice were then bred to produce F1 and F2 generations of ZIP8KO-microbiota mice. F1 ZIP8KO-microbiota mice were also infected with S. pneumoniae, and pulmonary host defense was assessed. Strikingly, the instillation of pneumococcus into the lung of F1 ZIP8KO-microbiota mice resulted in a significant increase in weight loss, inflammation, and mortality when compared to F1 wild-type (WT)-microbiota recipients. Similar defects in pulmonary host defense were observed in both genders, although consistently greater in females. From these results, we conclude that myeloid Zn homeostasis is not only critical for myeloid function but also plays a significant role in the maintenance and control of gut microbiota composition. Further, these data demonstrate that the intestinal microbiota, independent of host genetics, play a critical role in governing host defense in the lung against infection. Finally, these data strongly support future microbiome-based interventional studies, given the high incidence of zinc deficiency and the rs13107325 allele in humans.

Keywords: gut-lung axis; host defense; microbiome; pneumonia; zinc transporter.

Figure 1

Microbiota differences between ZIP8KO and WT mice are maintained in the first generation of mice following colonization of germ-free breeders. Composition of cecal microbiota from F0 and F1 mice was analyzed by 16s rRNA sequencing. β-diversity demonstrated by PCoA of Bray–Curtis dissimilarity in (A) male F0 and F1 mice with WT and ZIP8KO microbiota, (B) female F0 and F1 mice with WT and ZIP8KO microbiota, and (C) both male and female F0 and F1 mice with WT and ZIP8KO microbiota. (D) α-diversity determined by Chao1 index. (E) Taxonomic comparison of microbiota. (F) Differentially abundant ASVs as determined by LefSe using the Kruskal–Wallis rank sum test, followed by Linear Discriminant analysis.

Figure 2

Microbiota differences are maintained in the second generation of colonized mice. Composition of cecal microbiota from F2 mice was analyzed by 16s rRNA sequencing. β-diversity demonstrated by PCoA of Bray–Curtis dissimilarity in (A) male and female F2 mice with WT and ZIP8KO microbiota, (B) α- diversity determined by the Chao1 index. (C) Taxonomic comparison of microbiota. (D) Differentially abundant ASVs as determined by LefSe using the Kruskal–Wallis rank sum test, followed by Linear Discriminant analysis.

Figure 3

Myleoid-specific loss of Zip8 alters GI goblet cell numbers and composition. (A) Representative 20× images of PAS/AB-stained colonic cross-sections from male WT and ZIP8KO mice. (B) Number of PAS/AB+ goblet cells/10 crypts. (C,D) Quantification of the percentage of PAS/AB-stained areas (white arrows) and only AB-stained regions of each section (red arrows). (E) Colon length. Bars represent the mean ± SEM, and dots represent individual mice. p-values indicated in the figures were determined by Welch’s t-test.

Figure 4

ZIP8-associated dysbiosis contributes to alterations in GI goblet cell numbers and composition. (A) Representative 20× images of PAS/AB-stained colonic cross-sections from male F1 WT and F1 ZIP8KO-microbiota mice. (B) Number of PAS/AB + goblet cells/10 crypts. (C,D) Quantification of the percentage of PAS/AB-stained areas (white arrows) and only AB areas in each section (red arrows). (E) Colon length. (F) Serum FABP1 measurement of combined genders. Bars represent the mean ± SEM, and dots represent individual mice. p-values are indicated in the figure and were determined by Welch’s t-test.

Figure 5

ZIP8-associated dysbiosis contributes to alterations in lung Zn levels. Zn levels in (A) feces, (B) serum, and (C) lung tissue. Bars represent the mean ± SEM, and dots represent individual mice. p-values are indicated in the figure and were determined by Welch’s t-test.

Figure 6

ZIP8-associated dysbiosis increases pulmonary immune cell numbers. F1-microbiota mice were infected with S. pneumoniae, and the number of BALF immune cells was assessed. (A) Protein levels from BALF. (B) Total BAL counts. (C) Total macrophages post-infection. (D) Total neutrophils post-infection. Bars represent the mean ± SEM, and dots represent individual mice. p-values are indicated in the figure and were determined by Welch’s t-test.

Figure 7

ZIP8-associated dysbiosis increases pulmonary inflammation. F1-microbiota mice were infected with S. pneumoniae, and the levels of BALF cytokines were assessed. (A) IL-6 levels from BALF. (B) TNFα levels from BALF. (C) IL-10 levels from BALF. (D) CXCL1 levels from BALF. (E) IL-1β levels from BALF. Bars represent the mean ± SEM, and dots represent individual mice. p-values are indicated in the figure and were determined by Welch’s t-test.

Figure 8

ZIP8-associated dysbiosis increases pulmonary damage, as well as pulmonary bacterial burden and dissemination. F1-microbiota mice were infected with S. pneumoniae, and pulmonary damage and bacterial burden were assessed. (A) Lung inflammatory scores via H&E histology. (B) Circulating levels of surfactant protein-D. Log transformation burden of S. pneumoniae in the (C) lungs and (D) spleen. Bars represent the mean ± SEM, and dots represent individual mice. p-values are indicated in the figure and were determined by Welch’s t-test.

Figure 9

ZIP8-associated dysbiosis increases mortality following S. pneumoniae infection. F1-microbiota mice were infected with S. pneumoniae, and survival was assessed for 7 days post-infection. Survival post-infection in (A) males, (B) females, and (C) combined male and female mice. Percent weight change in (D) males, (E) females, and (F) combined male and female mice post-infection. Dots represent the mean and SD per group (n = 29 WT F1 mice, and n = 17 ZIP8KO F1 mice). p-values are indicated in the figure and were determined by the Chi-square test for each day post-infection.

Figure 10

Increased pulmonary immune cells in ZIP8-associated dysbiosic mice persist for 7 days post-infection. F1-microbiota mice were infected with S. pneumoniae, and the number of BALF immune cells was assessed. (A) Protein levels from BALF. (B) Total BAL counts. (C) Total macrophages post-infection. (D) Total neutrophils post-infection. Bars represent the mean ± SEM, and dots represent individual mice.