Intranasal Poly-IC treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population

Abstract

Type I IFN has been demonstrated to have major regulatory effects on the outcome of bacterial infections. To assess the effects of exogenously induced type I IFN on the outcome of Mycobacterium tuberculosis infection, we treated pathogen-exposed mice intranasally with polyinosinic-polycytidylic acid condensed with poly-l-lysine and carboxymethylcellulose (Poly-ICLC), an agent designed to stimulate prolonged, high-level production of type I IFN. Drug-treated, M. tuberculosis-infected WT mice, but not mice lacking IFN-alphabeta receptor 1 (IFNalphabetaR; also known as IFNAR1), displayed marked elevations in lung bacillary loads, accompanied by widespread pulmonary necrosis without detectable impairment of Th1 effector function. Importantly, lungs from Poly-ICLC-treated M. tuberculosis-infected mice exhibited a striking increase in CD11b+F4/80+Gr1int cells that displayed decreased MHC II expression and enhanced bacterial levels relative to the same subset of cells purified from infected, untreated controls. Moreover, both the Poly-ICLC-triggered pulmonary recruitment of the CD11b+F4/80+Gr1int population and the accompanying exacerbation of infection correlated with type I IFN-induced upregulation of the chemokine-encoding gene Ccl2 and were dependent on host expression of the chemokine receptor CCR2. The above findings suggest that Poly-ICLC treatment can detrimentally affect the outcome of M. tuberculosis infection, by promoting the accumulation of a permissive myeloid population in the lung. In addition, these data suggest that agents that stimulate type I IFN should be used with caution in patients exposed to this pathogen.

Figure 1. Poly-ICLC treatment increases host susceptibility to acute and chronic M. tuberculosis infection in mice.

WT mice were infected with M. tuberculosis (MTB) by aerosol and treated with Poly-ICLC, starting either 1 day (A–D) or 4 months (E and F) after exposure. (A) Representative images of H&E-stained lung sections and (B) histopathological evaluation of pulmonary necrosis in Poly-ICLC– or PBS-treated mice, 4 weeks after initiation of treatment of acutely infected mice. (C) Acid-fast staining and (D) bacterial load in the lungs of Poly-ICLC– or PBS-treated animals. (E) Survival and (F) pulmonary mycobacterial loads in Poly-ICLC– or PBS-treated mice (n = 5) exposed to M. tuberculosis 4 months earlier. The analysis was performed 3 weeks after treatment, due to increased mortality in drug-treated animals. Filled and open circles represent Poly-ICLC– or PBS-treated animals, respectively. In B, D, and F, circles indicate individual mice. Horizontal lines represent comparisons between Poly-ICLC– and PBS-treated, M. tuberculosis–infected mice. The data shown are representative of 5 (A–D) and 2 (E and F) independent experiments. Original magnification, ×50 (A); ×200 (C, first and third panels); ×400 (C, second and fourth panels). *0.01 < P value < 0.05, **P < 0.01.

Figure 2. Poly-ICLC–triggered exacerbation of murine tuberculosis is dependent on type I IFN signaling.

(A) Ifnb and (B) Ifna (all genes) mRNA expression levels, determined by real-time PCR in the lungs of M. tuberculosis–infected animals (open circles), naive mice treated with Poly-ICLC (gray circles), and M. tuberculosis–infected animals treated with Poly-ICLC (filled circles). Results are presented as the fold increase relative to transcript levels in PBS-treated, naive mice. Data are pooled from 3 independent experiments with similar results. (C) IFN-β protein levels determined by ELISA in lung homogenates of naive and M. tuberculosis–infected animals, PBS (open circles) or Poly-ICLC–treated (filled circles). (D) Mycobacterial loads in PBS-treated (open circles) or Poly-ICLC–treated (filled circles) WT and Ifnabr^–/– mice. (E) H&E-stained lung tissue sections demonstrating indistinguishable pulmonary pathology between Poly-ICLC– and PBS-treated, M. tuberculosis–infected Ifnabr^–/– mice. Circles indicate individual mice throughout. Results are representative of 3 independent experiments. Original magnification, ×25 (E). (A–D) Horizontal lines represent comparisons between Poly-ICLC– and PBS-treated, M. tuberculosis–infected mice. *0.01 < P value < 0.05, **P < 0.01.

Figure 3. Poly-ICLC treatment does not result in suppressed Th1 effector function in infected animals.

Naive or M. tuberculosis–infected WT mice were treated with PBS (open symbols) or Poly-ICLC (filled symbols) for 4 weeks, starting 1 day after pathogen exposure. (A) Frequency of CD4⁺CD44⁺ T cells in pulmonary leukocyte populations isolated from naive or M. tuberculosis–infected animals treated with PBS or Poly-ICLC. The data shown are the mean ± SEM (n = 5). (B) Frequency of T-bet⁺ CD4⁺ T cells, represented by horizontal lines, in the lungs of PBS- or drug-treated, infected animals. (C) Frequency of CD4⁺CD44⁺ T cells producing IFN-γ and TNF-α in response to in vitro stimulation with medium, anti-CD3, or PPD, determined by flow cytometry. Circles indicate individual animals. The data shown are representative of 3 independent experiments.

Figure 4. Poly-ICLC treatment triggers major alterations in pulmonary myeloid subsets in infected mice.

(A) Representative flow cytometry dot plots showing the frequencies and (B) bar graphs indicating the mean total numbers (± SEM) of CD11b⁺Gr1^–, CD11b⁺Gr1^int, and CD11b⁺Gr1^high pulmonary leukocytes isolated from naive or M. tuberculosis–infected animals treated with PBS (open bars) or Poly-ICLC (filled bars) (n = 5). The data shown are representative of 4 independent experiments. (C) Representative flow cytometry dot plots showing increased frequencies of F4/80-, CD115-, MAC-3-, and CD68-expressing cells in M. tuberculosis–infected mice after Poly-ICLC (bottom panels) or PBS administration (top panels). The data shown are representative of 3 independent experiments. (A and C) Numbers within or beside each box refer to the frequencies of cells within each gate.

Figure 5. The alterations in pulmonary myeloid cell composition triggered by Poly-ICLC in infected mice are dependent on type I IFN signaling.

Lung leukocytes were isolated from Poly-ICLC– and PBS-treated, M. tuberculosis–infected mice and analyzed by flow cytometry. (A) Representative dot plots and (B) bar graphs showing mean frequencies (± SEM) of CD11b⁺Gr1^int (top panel) and F4/80⁺MHCII⁺ (bottom panel) cells isolated from M. tuberculosis–infected WT mice (filled bars) or Ifnabr^–/– animals (open bars), treated with PBS or Poly-ICLC (n = 4–5). The data shown are representative of 2 independent experiments Numbers beside each box refer to the frequencies of cells within each gate. *0.01 < P value < 0.05.

Figure 6. The CD11b⁺Gr1^int subpopulation in the lungs of Poly-ICLC–treated mice displays impaired control of M. tuberculosis.

Pulmonary CD11b⁺Gr1^– and CD11b⁺Gr1^int cells were isolated by FACS from Poly-ICLC– and PBS-treated mice, 4 weeks after infection. The cells were then plated on agar, and bacterial CFUs were enumerated 3 weeks later. Bar graphs represent the mean ± SEM of the bacterial counts per 10⁵ lung cells recovered from multiple mice (n = 4–6). The experiment shown is representative of 3 performed. **P < 0.01.

Figure 7. Both recruitment of CD11b⁺Gr1^int cells and exacerbation of M. tuberculosis infection after Poly-ICLC treatment depend on CCR2 expression.

(A) Cxcl1, Cxcr2, and Ccl2 mRNA expression levels, determined by real-time PCR in the lungs of M. tuberculosis–infected animals treated with PBS (open circles) and Poly-ICLC (filled circles). The results shown represent the fold increase relative to that observed in untreated, naive mice, with circles representing individual mice. Data were pooled from 3 independent experiments with similar results. (B) Representative flow cytometry dot plots of CD11b⁺Gr1^–, CD11b⁺Gr1^int, and CD11b⁺Gr1^high pulmonary leukocytes isolated from Poly-ICLC– or PBS-treated, M. tuberculosis–infected WT mice or Ccr2-deficient animals. Numbers within or beside each box refer to the frequencies of cells within each gate. (C) Pulmonary mycobacterial loads in PBS-treated (open circles) and Poly-ICLC–treated (closed circles) WT and Ccr2-deficient mice. Circles represent individual animals. The data shown are representative of 3 independent experiments. (A and C) Horizontal lines represent comparisons between Poly-ICLC– and PBS-treated, M. tuberculosis–infected mice. **P < 0.01.