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. 2008 Jan 1;180(1):464-74.
doi: 10.4049/jimmunol.180.1.464.

Neutrophils clear bacteria associated with parasitic nematodes augmenting the development of an effective Th2-type response

John T Pesce  1 , Zhugong Liu, Hossein Hamed, Farhang Alem, Jeanette Whitmire, Hongxia Lin, Qian Liu, Joseph F Urban Jr, William C Gause
Affiliations

Neutrophils clear bacteria associated with parasitic nematodes augmenting the development of an effective Th2-type response

John T Pesce et al. J Immunol. .

Abstract

Infection with the parasitic nematode Nippostrongylus brasiliensis induces a potent Th2 response; however, little is known about early stages of the innate response that may contribute to protective immunity. To examine early events in this response, chemokine expression in the draining lymph node was examined after N. brasiliensis inoculation. Pronounced increases of several chemokines, including CCL2, were observed. Compared with wild-type mice, elevations in a Gr-1bright population in the draining lymph node was significantly decreased in CCL2-/- mice after N. brasiliensis inoculation. Further flow cytometric and immunofluorescent analysis showed that in wild-type mice, Gr-1+ cells transiently entered and exited the draining lymph node shortly after N. brasiliensis inoculation. The Gr-1bright population was comprised of neutrophils expressing TGF-beta and TNF-alpha. Following Gr-1+ cell depletion, N. brasiliensis infection resulted in transient, but significantly increased levels of IFN-gamma, increased serum IgG2a, reduced Th2 cytokines and serum IgE, greatly increased mortality, and delayed worm expulsion. Furthermore, bacteria were readily detected in vital organs. Infection of Gr-1+ cell-depleted mice with N. brasiliensis larvae that were pretreated with antibiotics prevented bacterial dissemination, Th1 inflammatory responses, and decreases in host survival. This study indicates that parasitic nematodes can be an important vector of potentially harmful bacteria, which is typically controlled by CCL2-dependent neutrophils that ensure the optimal development of Th2 immune responses and parasite resistance.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
CCL2-dependent Gr-1bright cell infiltration of the draining lymph node shortly after N. brasiliensis inoculation. A, BALB/c mice (5/treatment group) were intracutaneously inoculated with 500 N. brasiliensis L3. On days 1, 2, 3, or 6 after inoculation, draining CLN were removed, RNA purified, and analyzed for CCL2 mRNA using real-time quantitative fluorogenic RT-PCR. Fold changes are expressed relative to the untreated BALB/c control. B, CCL2KO and WT control mice (5/treatment group) were inoculated with N. brasiliensis L3 at the ear. Draining CLN were collected 18 h later. CLN cell suspensions were prepared and stained for anti-Gr-1-PE and anti-CD4-FITC. C, BALB/c WT mice were inoculated intracutaneously in one ear with N. brasiliensis L3. Draining CLN were removed at 18, 24, or 48 h after inoculation, and also from untreated control mice. CLN frozen sections were fluorescently stained with anti-B220-Alexa Fluor 647 (B cells; blue) and anti-Gr-1-FITC (green). Individual × 400 digital images were tiled together to form a single high resolution composite image of the draining lymph node. These experiments were repeated twice with similar results.
FIGURE 2
FIGURE 2
LN-infiltrating Gr-1bright cells are TNF-α- and TGF-μ-expressing neutrophils. A, BALB/c mice were inoculated with N. brasiliensis L3, and 18 h later CLN cells were harvested and stained with anti-Gr-1-PE, F480-FITC, anti-CD11b-PerCP-CY5.5, anti-CD11c-allophycocyanin, anti-CCR3-Alexa Fluor 647, or anti-DX5-allophycocyanin. B, In separate experiments, BALB/c mice (30/treatment group) were inoculated with N. brasiliensis L3, and 18 h later draining CLN were removed. Pooled cell suspensions were stained with anti-Gr-1 Ab. Gr-1bright and dull cells were isolated using high speed cell sorting (FACS). Sorted cells were centrifuged on slides using a Cytospin and then stained with DiffQuick to analyze morphology. C, RNA was purified from sorted cell populations, and real-time quantitative fluorogenic RT-PCR was used to assess cytokines and IL-4R gene expression. Sorted CD11c+ cells from untreated BALB/c mice were used as controls, and data are expressed as treated Gr-1+ cells/untreated CD11c+ cells. These experiments were repeated twice with similar results.
FIGURE 3
FIGURE 3
In the absence of Gr-1bright neutrophils, N. brasiliensis infection resulted in the rapid activation of immune cells and significantly increased IFN-γ expression. BALB/c mice were treated with anti-Gr-1 Ab to deplete Gr-1bright neutrophils, or given control Ig iso-type. One day later, the mice were inoculated in the ear with N. brasiliensis L3. The draining CLN were collected 18 h later. A, Single-cell suspensions were stained with anti-B220, anti-CD11c, and anti-CD4 or anti-CD8 plus anti-CD69, anti-B7.2, anti-MHC II, or anti-CD40 Abs and analyzed by FACS. Expression of cell surface costimulatory molecules was assessed on gated B220+ B cells and CD11c+ dendritic cells. Expression of CD69 was examined on gated CD4 T cells and CD8 T cells. This experiment was repeated twice with similar results. B, In a separate experiment, the collected CLN were further processed for RNA isolation and analyzed for IFN-γ gene expression using real-time quantitative fluorogenic RT-PCR. All data are expressed as relative to that of untreated controls, and the mean and SE are shown for each treatment group (5 mice/group). This experiment was repeated three times with similar results. C, BALB/c mice were administrated either with anti-Gr-1, anti-TGF-β, anti-TNF-α, or anti-TGF-β plus anti-TNF-α Abs, and 1 day later inoculated in the ear with N. brasiliensis L3. Gene expression of IFN-γ was analyzed by real-time quantitative fluorogenic RT-PCR. This experiment was repeated twice with similar results.
FIGURE 4
FIGURE 4
Presence of Gr-1bright neutrophils promotes survival, wound healing, and host-protective Th2-type responses. BALB/c mice (5–10 mice/group) were injected with depleting anti-Gr-1 or isotype control Abs. One day later, these mice were inoculated in the ear with N. brasiliensis L3. Treated mice were monitored closely on a daily basis. A, Approximately 60–80% of Gr-1 cell-depleted mice were killed by N. brasiliensis infection on ~day 2 or 3. This experiment was repeated five times with similar results. B, A delayed-type hypersensitivity response developed at the infection site (ear) starting from day 2 postinfection in Gr-1 cell-depleted mice. This experiment was repeated five times with highly reproducible results. C, On day 7 postinfection, mesenteric lymph nodes were collected from surviving mice. RNA was purified and quantitative fluorogenic real-time RT-PCR was used to assess cytokine gene expression. This experiment was repeated three times with similar results. D, In a separate experiment, BALB/c mice were similarly treated and, 10 days post-N. brasiliensis inoculation, adult worms and egg production were determined. E, Total serum IgG2a and IgE levels were determined by ELISA. The mean and SE derived from five individual mice are shown for each treatment group. Graphs are representative of three individual experiments with similar results.
FIGURE 5
FIGURE 5
N. brasiliensis-infected CCL2KO mice showed increased Th1 response and suppressed host protectionA, CLN IL-4 and IFN-γ gene expression at 18 h after N. brasiliensis L3 inoculation was determined by real-time quantitative fluorogenic RT-PCR. B, In a separate experiment, tissue samples were collected 10 days post-N. brasiliensis infection, and serum IgG2a levels and egg production were assessed. This experiment was performed three times.
FIGURE 6
FIGURE 6
Infection with antibiotic-treated L3 blocked the effects of neutrophil depletion following N. brasiliensis inoculation. N. brasiliensis L3 were incubated with antibiotics (400 U of penicillin, 400 μg/ml streptomycin, and 400 μg/ml neomycin) or PBS for 2 h, and then washed with PBS three times. A, Treated and untreated N. brasiliensis L3 were placed on blood agar plate and cultured at 37°C for 24 h. This experiment was repeated three times with similar results. B, BALB/c mice (5/treatment group) were administered anti-Gr-1 Ab i.p., and 1 day later inoculated with antibiotic-treated or untreated N. brasiliensis L3. IFN-γ gene expression was assessed by real-time quantitative fluorogenic RT-PCR at 18 h after inoculation. C, In separate experiments, neutrophil-depleted mice were inoculated with antibiotic-treated or untreated N. brasiliensis L3, and the survival rate after N. brasiliensis infection was recorded. D, Delayed-type hypersensitivity developed at the infection site (right ear) of untreated, but not antibiotic-treated N. brasiliensis-inoculated neutrophil-depleted mice. E, On day 7 after infection, mesenteric lymph nodes were collected from surviving mice, and IL-4 and IL-13 mRNA levels were assessed by real-time quantitative fluorogenic RT-PCR. The mean and SE derived from five individual mice are shown for each treatment group. These experiments were repeated twice with similar results.
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References

    1. Gause WC, Urban JF, Stadecker MJ. The immune response to parasitic helminths: insights from murine models. Trends Immunol. 2003;24:269–277. - PubMed
    1. Ogilvie BM, Hockley DJ. Effects of immunity of Nippostrongylus brasiliensis adult worms: reversible and irreversible changes in infectivity, reproduction, and morphology. J Parasitol. 1968;54:1073–1084. - PubMed
    1. Liu Z, Liu Q, Pesce J, Whitmire J, Ekkens MJ, Foster A, VanNoy J, Sharpe AH, Urban JF, Jr, Gause WC. Nippostrongylus brasiliensis can induce B7-independent antigen-specific development of IL-4-producing T cells from naive CD4 T cells in vivo. J Immunol. 2002;169:6959– 6968. - PubMed
    1. Trujillo-Vargas CM, Mayer KD, Bickert T, Palmetshofer A, Grunewald S, Ramirez-Pineda JR, Polte T, Hansen G, Wohlleben G, Erb KJ. Vaccinations with T-helper type 1 directing adjuvants have different suppressive effects on the development of allergen-induced T-helper type 2 responses. Clin Exp Allergy. 2005;35:1003–1013. - PubMed
    1. Liu Z, Liu Q, Hamed H, Anthony RM, Foster A, Finkelman FD, Urban JF, Jr, Gause WC. IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites. J Immunol. 2005;174:2242–2249. - PMC - PubMed

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