Development of tolerogenic dendritic cells and regulatory T cells favors exponential bacterial growth and survival during early respiratory tularemia

Abstract

Tularemia is a vector-borne zoonosis caused by Ft, a Gram-negative, facultative intracellular bacterium. Ft exists in two clinically relevant forms, the European biovar B (holarctica), which produces acute, although mild, self-limiting infections, and the more virulent United States biovar A (tularensis), which is often associated with pneumonic tularemia and more severe disease. In a mouse model of tularemia, respiratory infection with the virulence-attenuated Type B (LVS) or highly virulent Type A (SchuS4) strain engenders peribronchiolar and perivascular inflammation. Paradoxically, despite an intense neutrophilic infiltrate and high bacterial burden, T(h)1-type proinflammatory cytokines (e.g., TNF, IL-1β, IL-6, and IL-12) are absent within the first ∼72 h of pulmonary infection. It has been suggested that the bacterium has the capacity to actively suppress or block NF-κB signaling, thus causing an initial delay in up-regulation of inflammatory mediators. However, our previously published findings and those presented herein contradict this paradigm and instead, strongly support an alternative hypothesis. Rather than blocking NF-κB, Ft actually triggers TLR2-dependent NF-κB signaling, resulting in the development and activation of tDCs and the release of anti-inflammatory cytokines (e.g., IL-10 and TGF-β). In turn, these cytokines stimulate development and proliferation of T(regs) that may restrain T(h)1-type proinflammatory cytokine release early during tularemic infection. The highly regulated and overall anti-inflammatory milieu established in the lung is permissive for unfettered growth and survival of Ft. The capacity of Ft to evoke such a response represents an important immune-evasive strategy.

Figure 1.. Ft induces activation of NF-κB signaling.

RAW264.7 cells (5×10⁵/ml) were unstimulated or stimulated for 6 h with Ft LVS (MOI of 100), LPS (100 ng), Pam3CSK (100 ng), or purified rTul4 lipoprotein (100 ng). Ft was grown in liquid culture (i.e., MHB or BHI broth) or isolated from infected MΦ, as described in Materials and Methods. Following stimulation, cells were analyzed by flow cytometry for eGFP expression. To confirm activation of NF-κB (as reflected in induced GFP expression), one set of cells was stimulated in the presence of the IKK inhibitor PTL (30 μM). FACS results are representative of three independent experiments. SSC, Side-scatter.

Figure 2.. The inability of Ft to elicit TNF production does not reflect induction of tolerance, SOCS activity, or alternative activation of MΦ.

(A) BMDMs (5×10⁵ cells/ml) were exposed to a low-dose primary (1°) stimulus of BHI-grown Ft LVS (MOI of 10), LPS (100 ng/ml), or BLP (100 ng/ml) for 4 h at 37°C. Cells were then washed twice and re-exposed to a higher homologous or heterologous secondary (2°) stimulus, and TNF levels were measured after 24 h. The values are expressed as mean ± sem from two independent experiments. (B and C) BMDMs (5×10⁵ cells/ml) were infected with non-HAd (i.e., MHB-gown) or HAd (i.e., BHI- or MΦ-grown) Ft LVS (MOI of 100), and total RNA was recovered from cells after 1, 3, 6, and 24 h incubation. qPCR was used to determine transcript levels for sosc1, socs3, tnf, inos, and arg1. The values are expressed as mean ± sem from three independent experiments. *P < 0.05; ***P < 0.001.

Figure 3.. TLR2 is required for Ft LVS- and SchuS4-induced anti-inflammatory cytokine release from DCs.

DCs (5×10⁵ cells/ml) from WT and TLR2^−/− mice were infected with MHB-, BHI-, and MΦ-grown Ft LVS (A and B) or MHB- and BHI-grown SchuS4 (C; MOI of 100 for each) for 24 h; culture supernatants were analyzed for proinflammatory cytokines by CBA; and anti-inflammatory cytokines were measured using commercial ELISA. The values are expressed as mean ± sem from three independent experiments. *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 4.. Ft induces primarily anti-inflammatory cytokines during early respiratory infection.

Four groups of C57BL/6 mice were i.n.-infected with 1 × 10³ CFU of BHI-grown Ft LVS and were killed at Days 1, 3, 5, and 7 PI. One group of sham-inoculated mice served as a control. The lung homogenates prepared from the uninfected lungs and infected lungs were analyzed for proinflammatory (A–C) and anti-inflammatory (D) cytokines. The values are expressed as mean ± sem from four independent experiments (n=14 total mice/group). *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 5.. Myeloid and lymphoid cells are a source of anti-inflammatory cytokines during early respiratory infection.

Two groups of C57BL/6 mice were i.n.-infected with 1 × 10³ CFU of BHI-grown Ft LVS and killed on Days 2 and 6 PI. One group of sham-inoculated mice served as a control. (A) Histological evaluation of the lungs at Days 2 and 6 PI. Original magnification is ×200 or ×1000, oil immersion. Arrowheads, Polymorphonuclear cells; arrows, mononuclear cells; and *, multinucleated giant cells. (B–E) Lung cells were analyzed for ICCS by flow cytometry. The percentages neutrophils, naMΦ, DCs, and CD3⁺ T cells expressing TNF, IL-10, or TGF-β are shown. The values are expressed as mean ± sem from two independent experiments (n=11 total mice/group). *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 6.. IL-17-producing cells were activated rapidly in the lungs of Ft-infected mice.

Four groups of C57BL/6 mice were i.n.-infected with 1 × 10³ CFU of BHI-grown Ft LVS and killed at Days 1, 3, 5, and 7 PI. One group of sham-inoculated mice served as a control. (A) Levels of IL-17A were measured in the lung homogenates by ELISA. (B–F) Lung cells were analyzed for ICCS of IL-17A by flow cytometry. The percentages and total numbers of different cell types producing IL-17A are shown. The values are expressed as mean ± sem from four independent experiments (n=14 total mice/group). *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 7.. Ft induces the development of pulmonary tDCs and T_regs.

Two groups of C57BL/6 mice were infected with 1 × 10³ CFU of BHI-grown Ft LVS and killed at Days 2 and 6 PI. One group of sham-inoculated mice served as a control. (A) CD11c^high lung cells were evaluated by flow cytometry for surface expression of the tDC markers, CD103 and CCR9. CD11c^highCD103⁺ cells (B) and CD11c^highCCR9⁺ cells (C) were analyzed for IL-10 and TGF-β cytokine production by flow cytometry. (D) T_regs were identified on the basis of FoxP3 expression. Gating on CD4⁺ cells, the percentages, and total numbers of CD25⁺FoxP3⁺ cells were calculated. The values are expressed as mean ± sem from four independent experiments (n=14 total mice/group). *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 8.. Levels of TGF-β, numbers of pulmonary T_regs, and Ft burden are interrelated features of early tularemia pathogenesis.

(A) Four groups of C57BL/6 mice were i.n.-infected with 1 × 10³ CFU of BHI-grown Ft LVS and killed at Days 1, 3, 5, and 7 PI. One group of sham-inoculated mice served as a control. Bacterial burden was determined by colony plating and presented as log₁₀ CFU. (B–D) Points represent individual animals, and lines are linear regression fits to data for each day. (B) The bacterial burden is positively correlated with TGF-β levels on Days 1 [r²=0.67; P=0.013; log(CFUs)=3.79+137(10⁻⁶)×TGF-β] and 3 [r²=0.77; P<0.01; log(CFUs)=6.37+82(10⁻⁶)×TGF-β]. (C) The bacterial burden is positively correlated with numbers of T_regs on Days 1 [r²=0.65; P=0.02; log(CFUs)=4.10+314(10⁻⁶)×T_regs] and 3 [r²=0.71; P<0.01; log(CFUs)=6.84+58(10⁻⁶)×T_regs]. (D) Numbers of T_regs are positively correlated with TGF-β levels at Days 0 (r²=0.91; P<0.001; T_regs=–231+0.254×TGF-β), 1 (r²=0.71; P<0.01; T_regs=–524+0.362×TGF-β), and 3 (r²=0.75; P<0.01; T_regs=–5982+1.17×TGF-β).