Murine Respiratory Tract Infection with Classical Klebsiella pneumoniae Induces Bronchus-Associated Lymphoid Tissue

Abstract

Klebsiella pneumoniae is a Gram-negative, opportunistic pathogen that commonly causes nosocomial pneumonia, urinary tract infection, and septicemia. Our recent work utilizing a murine model of respiratory tract infection with classical K. pneumoniae demonstrated leukocyte aggregates in the lungs of mice at 28 days postinfection. Here, we sought to characterize the composition and development of these structures. Histopathological analyses of murine lungs revealed immune cell clusters surrounding the pulmonary vasculature and airways by 14 days postinfection, resembling inducible bronchus-associated lymphoid tissue (iBALT). Further investigation of these structures demonstrated central B cell aggregates with concomitant dispersed T cells. At day 28 postinfection, these lymphoid clusters expressed germinal center markers and CXCL12, qualifying these structures as iBALT with nonclassical B cell follicles. Investigations in mutant mice revealed that those lacking B and/or T cells were not able to form fully defined iBALT structures, although some rudimentary B cell clusters were identified in mice lacking T cells. The longevity of K. pneumoniae-induced BALT was assessed for up to 120 days postinfection. Lymphoid aggregates significantly decreased in size and quantity by 90 days after K. pneumoniae infection; however, aggregates persisted in mice that were restimulated with K. pneumoniae every 30 days. Finally, infections of mice with an array of classical K. pneumoniae clinical isolates demonstrated that the development of these structures is a common feature of K. pneumoniae lung infection. Together, these data confirm that murine lungs infected with K. pneumoniae develop iBALT, which may play a role in pulmonary immunity to this troublesome pathogen.

Keywords: B cells; Klebsiella pneumoniae; T cells; iBALT; induced bronchus-associated lymphoid tissue; pneumonia.

FIG 1

Lymphoid aggregate development in mice infected with K. pneumoniae. (A) H&E staining of left lung sections at 0, 7, 14, 21, and 28 dpi, including a higher magnification of the day 28 aggregate (inset and bottom right). (B) Average blindly assigned lymphocytic aggregate scores with standard deviations (score of 3 assigned for all mice at 21 dpi). Images are representative, and scores are averages from 4 to 7 mice evaluated per time point. Bars, 100 μm.

FIG 2

Characterization of lymphoid aggregate development and composition in mice infected with K. pneumoniae. (A) B cell (B220) and T cell (CD3) IHC staining of murine lungs at 7, 14, 21, and 28 dpi (and the PBS control). (B) Germinal center B cell (PCNA) (top) and conventional dendritic cell (CD11c) (bottom) IF staining of lymphoid aggregates in mice at 28 dpi. (C) CXCL12 IF staining of lymphoid aggregates in mice at 28 dpi. Images are representative of results from 4 to 6 mice evaluated per time point. Bars, 20 μm.

FIG 3

Evaluation of lymphoid aggregate formation in mutant mice infected with K. pneumoniae. (A) H&E staining of murine left lung sections at 28 dpi in wild-type (WT), Rag1^−/−, μMT^−/−, and TCRβ⁻/δ⁻ mice. (B) B cell (B220) and T cell (CD3) IHC staining of murine lungs at 28 dpi in WT, μMT^−/− and TCRβ⁻/δ⁻ mice. Images are representative of results from 3 to 6 mice evaluated per strain. Bars, 100 μm (A) and 20 μm (B).

FIG 4

Lymphocytic aggregate durability with and without K. pneumoniae reinfection. (A) H&E staining of left lung sections 60, 90, and 120 days following either a single K. pneumoniae infection at day 0 (top) or repeat infections every 30 days (bottom). (B) Average blindly assigned lymphocytic aggregate scores with standard deviations. Images are representative, and scores are averages from 4 to 6 mice evaluated per time point. Bars, 100 μm. ns, not significant; *, P value of <0.05.

FIG 5

iBALT formation in response to infection with distinct K. pneumoniae clinical isolates. Shown is H&E staining of left lung sections 28 days following mock infection (PBS) or infection with a classical K. pneumoniae lower respiratory isolate (KR3, KR45, KR74, KR122, or KR174). Images are representative of results from 2 to 5 mice evaluated. Bars, 100 μm.