Critical role for interleukin-1beta (IL-1beta) during Chlamydia muridarum genital infection and bacterial replication-independent secretion of IL-1beta in mouse macrophages

Abstract

Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection. In the present study, IL-1beta(-/-) mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1beta both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1beta secretion in WT mice, the major producers of IL-1beta in vivo are F4/80(+) macrophages and GR-1(+) neutrophils, but not CD45(-) epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1beta, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1beta from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1beta secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1beta secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1beta secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1beta mRNA observed in vivo in this cell type.

FIG. 1.

IL-1β deficiency delays chlamydial clearance and reduces oviduct pathology. (A) C. muridarum infection course in WT (n = 10) and IL-1β KO (n = 9) mice. Log₁₀ numbers of IFU per milliliter were calculated as described in Materials and Methods and graphed as means ± standard errors of the means (SEM). Significance (P = 0.002) was determined by two-way RM ANOVA and is denoted by asterisks. Numbers next to datum points indicate the fraction of animals infected at the particular time point. (B) Percent hydrosalpinx among WT (n = 20) and IL-1β KO (n = 16) oviducts (2 oviducts/mouse) harvested 42 days postinfection. The asterisk indicates a significant difference (P < 0.05) in the proportion of oviducts with hydrosalpinx as determined by the Fisher exact test. (C) The median group pathology score for each category of cells from oviducts harvested from WT and IL-1β KO mice 42 days postinfection is graphed. The asterisk denotes a statistically significant difference (P < 0.05) in oviduct dilation between the two groups as determined using Friedman RM ANOVA by ranks, followed by Student-Newman-Keuls multiple-comparison procedure. The distribution of individual dilation scores is also shown. PMNs, polymorphonuclear leukocytes; lymphs/mono, lymphocytes/monocytes.

FIG. 2.

Macrophages and neutrophils are the principal source of IL-1β in vivo. (A) Genital tract secretions from C57BL6/J mice (n = 5) infected with C. muridarum (3 × 10⁵ IFU/mouse) were isolated and analyzed for IL-1β protein. Data are means ± standard errors of results representative of those from three independent experiments. Asterisks denote significant differences (P < 0.05 by one-way RM ANOVA) from day 0 baseline levels. (B) Percent CD45⁺ cells in the cervices of mice (n = 3) at day 0 (d0; preinfection) and day 4 (d4) postinfection with 3 × 10⁵ IFU of C. muridarum/mouse. The asterisk denotes a P value of <0.05 by the t test. (C and D) Populations of CD45⁻ epithelial cells, CD45⁺ Gr-1⁺ F4/80⁺ macrophages, and CD45⁺ Gr-1⁺ F4/80⁻ neutrophils before (C) and after (D) sorting are depicted. Values represent percentages of cells within the indicated gate. The infection dose was 3 × 10⁶ IFU/mouse. CD45-PerCP5.5, peridinin chlorophyll protein-Cy5.5 CD45 antibody; Gr1-FITC, fluorescein isothiocyanate Gr-1 antibody; F4/80-APC, allophycocyanin F4/80 antibody; CD45-ve, CD45-positive; PMN, polymorphonuclear leukocytes. (E and F) RNAs and cellular lysates from CD45⁻, macrophage (Macs), and neutrophil (PMN) cell populations were analyzed for IL-1β mRNA (E) and intracellular IL-1β protein (F) expression. IL-1β mRNA levels were normalized with respect to actin gene expression, while IL-1β protein levels in cell lysates were normalized with respect to the cell number (results are expressed in picograms per 10⁵ cells). (G) The same RNA samples and RNA from an uninfected (UI) mouse cervix were assayed for chlamydial rs16 RNA. Data in panels E, F, and G are means ± standard deviations of results for samples assayed in duplicate. Results from one of two similar experiments are shown.

FIG. 3.

TLR prestimulation of macrophages enhances chlamydia-induced IL-1β secretion. (A) Peritoneal macrophages were infected in vitro with live or heat-killed C. muridarum, and the upregulation of IL-1β mRNA at 3, 8, and 24 h postinfection was monitored. Data are mean levels of IL-1β cDNA ± standard deviations for samples assayed in duplicate in a representative experiment. (B) Macrophages were pretreated with either 100 ng/ml E. coli LPS or medium alone for 6 h before infection with viable, UV-inactivated (UV inactiv), or heat-killed C. muridarum. Data are means ± standard deviations of results for samples assayed in duplicate in a representative experiment. (C) Macrophages were prestimulated with increasing doses of C. trachomatis L2 LPS and then infected with C. muridarum as described in the legend to panel B. The asterisk denotes a significant difference (P < 0.05 by one-way ANOVA) from levels in infected, unprimed cells. (D) Peritoneal macrophages were prestimulated with either 25 μg/ml of the TLR3 ligand poly(I:C) or 2 μg/ml of the TLR2 ligand Pam3CSK4 and infected with C. muridarum. For analyses in panels B, C, and D, supernatants were harvested at 18 h postinfection and assayed for IL-1β. Double and triple asterisks denote significant differences (**, P = 0.025, and ***, P = 0.002 by an unpaired t test) between primed and unprimed cells. Data in panels C and D are means ± standard deviations of values obtained from three independent experiments.

FIG. 4.

IL-1β secretion from primed macrophages requires caspase-1, potassium efflux, and the activity of serine proteases. (A) E. coli LPS-prestimulated (primed) macrophages were treated with 50 μM Ac-YVAD-CHO and 50 μM TPCK individually or in tandem, 10 μM MG-132, or vehicle (DMSO) alone and then infected with C. muridarum as described in Materials and Methods. (B) E. coli LPS-prestimulated macrophages were treated with 130 mM KCl or 5 mM glycine prior to infection. Asterisks in panels A and B denote significant differences (*, P < 0.05, and **, P < 0.01 by one-way ANOVA) from results for LPS-primed macrophages infected with C. muridarum. (C) E. coli LPS-primed macrophages were treated with 5 mM ATP or infected with C. muridarum in the presence or absence of 2.5 U/ml of apyrase. The asterisk denotes a significant difference (P = 0.016 by unpaired t test) between ATP-induced IL-1β secretion levels in the presence and absence of apyrase. For all experiments, supernatants were harvested at 18 h postinfection and assayed for IL-1β. Data in all panels are means ± standard deviations of values obtained from three independent experiments.

FIG. 5.

IL-1β secretion from primed macrophages requires chlamydial viability but not growth. (A) E. coli LPS-primed macrophages were infected with either heat-killed C. muridarum (Cm) or viable C. muridarum or C. caviae. Additionally, certain macrophages receiving live C. muridarum were also treated with either 150 μg/ml rifampin (Rif) or 50 μM INP0007. Supernatants were harvested at 18 h postinfection and assayed for IL-1β. Data are means ± standard deviations of values obtained from three independent experiments. Asterisks denote significant differences (P < 0.05 by one-way ANOVA) from the results for LPS-only treatment. (B) Staining for chlamydial inclusions in all the treated groups described in the legend to panel A.

FIG. 6.

Inhibition of phagocytic uptake of C. muridarum does not impair IL-1β secretion. E. coli LPS-primed macrophages were infected following pretreatment with increasing doses of cytochalasin D. Chlamydial rs16 expression from cellular RNA (A) and IL-1β levels in culture supernatants (B) were assayed. Error bars in panel A represent means ± standard deviations of results for samples assayed in duplicate in a single experiment, while error bars in panel B represent means ± standard deviations of values obtained from two independent experiments.

FIG. 7.

Cellular lysates containing heat-killed chlamydial RBs are sufficient to prestimulate macrophages. Macrophages were prestimulated for 6 h with lysates from C. muridarum-infected or uninfected (UI) HeLa cells. 1X and 10X correspond to lysates derived from 2.5 × 10⁴ and 2.5 × 10⁵ cells, respectively. Where indicated, the lysates were subjected to heat treatment for heat killing (HK) of any viable RBs/EBs. (A) After prestimulation, macrophages were infected with C. muridarum and IL-1β protein levels were assayed at 18 h postinfection. (B) In parallel, IL-1β mRNA in macrophages was also quantitated after the initial priming step and immediately prior to infection. Error bars in panels A and B represent means ± standard deviations of results for samples assayed in duplicate.