Enteric pathogens induce tissue tolerance and prevent neuronal loss from subsequent infections

Abstract

The enteric nervous system (ENS) controls several intestinal functions including motility and nutrient handling, which can be disrupted by infection-induced neuropathies or neuronal cell death. We investigated possible tolerance mechanisms preventing neuronal loss and disruption in gut motility after pathogen exposure. We found that following enteric infections, muscularis macrophages (MMs) acquire a tissue-protective phenotype that prevents neuronal loss, dysmotility, and maintains energy balance during subsequent challenge with unrelated pathogens. Bacteria-induced neuroprotection relied on activation of gut-projecting sympathetic neurons and signaling via β₂-adrenergic receptors (β2AR) on MMs. In contrast, helminth-mediated neuroprotection was dependent on T cells and systemic production of interleukin (IL)-4 and IL-13 by eosinophils, which induced arginase-expressing MMs that prevented neuronal loss from an unrelated infection located in a different intestinal region. Collectively, these data suggest that distinct enteric pathogens trigger a state of disease or tissue tolerance that preserves ENS number and functionality.

Keywords: enteric infections; enteric neurons; eosinophils; macrophages; neuroimmunology; small intestine.

Figure 1.. Y. pseudotuberculosis infection induces neuronal protection during subsequent infection.

(A-D) C57BL/6J mice were orally gavaged with PBS (non-infected, NI), 10⁹ colony-forming units (CFU) of Salmonella Typhimurium spiB, 10⁸ CFU of Y. pseudotuberculosis (Yp) only or 10⁸ CFU of Yp and 21 days later with 10⁹ CFU of spiB. (A) Quantification of fecal Yp CFU. (B) Neuronal quantification in the ileum myenteric plexus assessed by IF staining of ANNA-1 on day 10 post-spiB infection. Left, representative images; Scale bars, 50 μm. (C) Quantification of fecal spiB CFU. (D) Total GI transit time measured at 12 days post infection (dpi). (E) C57BL/6J mice were orally gavaged with PBS, spiB or Yp. At 20 dpi Yp-infected mice were treated with anti-CSF1R or IgG isotype control, followed by oral gavage with spiB. Ileum myenteric plexus neurons were quantified at 7 dpi. (F and G) Flow cytometry analysis of Arg1-expressing macrophages isolated from ileum muscularis at 14 dpi with Yp or spiB. (H) Quantification of ileum myenteric plexus neurons of LysM^ΔArg1 mice and WT littermates orally gavaged with Yp only or followed by spiB infection 21 days later. (I-K) LysM^ΔAdrb2 (Cre+) mice and WT (Cre−) littermates were orally gavaged with Yp and 21 days later with spiB. (I) Neuronal quantification of ileum myenteric plexus 7 days post-spiB infection. (J) Arg1 expression by macrophages in ileum muscularis at 7 dpi as determined by flow cytometry. (K) Total gastrointestinal (GI) transit time at 12 dpi. Dashed lines indicate the range of day 7 enteric neuron numbers defined by mean ± SEM of a set of control non-infected C57BL/6J mice (n=25). Data is from 2 pooled independent experiments (3–5 mice per condition, A-C and F) or one out of two representative experiments (D-E and G-K). See also Figure S1.

Figure 2.. S. venezuelensis infection induces long-term neuronal protection during subsequent infection.

(A-D) C57BL/6J mice were s.c. injected with water and then orally gavaged with PBS (non-infected, NI) or infected with spiB, Sv only or Sv and 14 days later with spiB. (A) Quantification of fecal Sv eggs. (B) Right, Neuronal quantification in the ileum myenteric plexus assessed by IF staining using ANNA-1 on day 8 post- spiB infection or 14 days post-Sv infection. Left, representative images; Scale bars, 50 μm. (C) Quantification of fecal spiB CFU. (D) Total GI transit time measured at 10 dpi. (E) C57BL/6J mice were orally gavaged with PBS or infected with spiB or Sv. At 14 dpi Sv-infected mice were treated with anti-CSF1R or IgG isotype control, followed by oral gavage with spiB. Ileum myenteric plexus neurons were quantified 7 days post-spiB infection. (F) Flow cytometry analysis of Arg1-expressing macrophages isolated from ileum muscularis at 14 dpi with Sv or spiB. (G) Neuronal quantification of ileum myenteric plexus of LysM^ΔArg1 (Cre+) mice and WT (Cre−) littermates orally gavaged with Sv only or followed by spiB infection 14 days later. (H) Number of cFos+ neurons in CG-SMG 3 days after Yp infection, 7 days after Sv infection and in non-infected controls. (I-K) LysM^ΔAdrb2 mice and WT littermates were orally gavaged with Sv and 14 days later with spiB. (I) Quantification of ileum myenteric plexus neurons 7 days post-spiB infection. (J) Arg1 expression by macrophages in ileum myenteric plexus at 7 dpi. (K) Total GI transit time at 10 dpi. (L and M) C57BL/6J mice were orally gavaged with spiB only or s.c. injected with Sv and 8, 12 or 24 weeks later infected with spiB. (L) Ileum myenteric plexus neurons were quantified 10 days post-spiB infection. (M) Arg1 expression by macrophages in ileum myenteric plexus at 7 dpi as determined by flow cytometry. (G and I) Dashed lines indicate the range of day 7 enteric neuron numbers defined by mean ± SEM of a set of control non-infected C57BL/6J mice (n=25). Data is from 2 pooled independent experiments (3–5 mice per condition, A-C and H) or one out of two representative experiments (D-G and I-M). See also Figure S2.

Figure 3.. Transcriptomic analysis of MMs.

(A-F) C57BL/6J mice were s.c. injected with water (non-infected, NI) or infected with spiB, Sv or Yp. Ileum MMs were sorted for RNA-seq at 14dpi. (A-D) Volcano plots showing comparisons between (A) Yp and spiB group, (B) Sv and spiB group, (C) Sv and Yp group and (D) infected (all infections pooled) and non-infected group. All significantly regulated genes (FDR<0.01) are colored. (E-F) GSEA of MM gene signature from all infected mice in indicated publicly available datasets. See also Table S1.

Figure 4.. Immune response to S. venezuelensis is associated with neuronal protection during subsequent infection.

(A-F) C57BL/6J mice were s.c. injected with water (non-infected, NI) or infected with spiB, Sv only or Sv and 14 days later with spiB. (A) Flow cytometric intranuclear analysis of CD4⁺ T cells in lamina propria (LP) expressing indicated transcription factors 2 days post-spiB infection. (B and C) Frequency of ileum (B) and duodenum (C) LP eosinophils at 14 dpi with Sv. (D) Frequency of duodenum LP eosinophils in mice infected with spiB only and Sv followed by spiB 12 or 24 weeks later and analyzed at 7 dpi. (E and F) Serum levels of IL-4 (E) and IL-13 (F) measured over time post-infection with spiB or Sv. (G) Experimental design for (H-K). (H-K) C57BL/6J mice were infected with spiB or Sv. On indicated days Sv-infected mice were treated with anti-CD4 antibody, followed by treatment with ivermectin and oral gavage with spiB. (H) Quantification of fecal Sv eggs. (I) Quantification of ileum myenteric plexus neurons 7 days post-spiB infection. Dashed lines indicate the range of day 7 enteric neuron numbers defined by mean ± SEM of a large set of control C57BL/6J mice. (J) Frequency of duodenum LP eosinophils at 7 dpi. (K) Arg1 expression by ileum myenteric plexus macrophages at 7 dpi. (L) C57BL/6J mice were subcutaneously injected Sv or PBS. IL-5 expression in CD4⁺ T cells was determined after 4-hour in vitro stimulation of duodenum LP cells with PMA and ionomycin at 21 dpi. Data in (C) is from 2 pooled independent experiments (3 mice per condition). All other data is from one representative out of two independent experiments with 3–4 mice per condition. See also Figure S3.

Figure 5.. Pet store mice are resistant to neuronal loss upon spiB infection.

Pet store mice were analyzed at steady-state (A-C and E-J) or 7 days post spiB-infection (A, C and D). (A) Quantification of ileum myenteric plexus neurons. (B) Small intestine length. (C) Number of neuron per ganglion within the ileum myenteric plexus. (D) Quantification of fecal spiB CFU. Shaded area indicates range of spiB CFU numbers defined by mean ± SEM from a large set of control SPF mice. (E) Frequency of duodenum LP eosinophils. (F) Serum levels of IL-4 and IL-13. (G) Frequency of Arg1-expressing ileum myenteric plexus macrophages. (H-J) Frequencies of indicated bone marrow progenitor cell subsets. (K) Frequency of Arg1-expressing ileum MMs in non-infected BALB/c and C57BL/6 mice. (L) BALB/c mice were orally gavaged with PBS (non-infected, NI) or spiB. Ileum myenteric plexus neurons were quantified at 15 dpi. Data is from one representative out of 2 independent experiments (B-J) or 2 pooled independent experiments with 3–5 mice per condition (A and K).

Figure 6.. IL-4 and IL-13 producing eosinophils mediate Sv-induced neuronal protection.

(A-C) iPHIL mice and WT littermates were infected with Sv and treated with DT on day 0, 1, 4, 6 and 8 post-infection. Analysis was performed at 10 dpi. (A and B) Frequency of duodenum (A) and ileum (B) LP eosinophils. (C) Quantification of fecal Sv eggs. (D-F) iPHIL mice and WT littermates were infected with Sv and treated with DT on day 0, 1, 4, 7, 10, 13, 16 and 18 post-infection. On day 14 post-Sv infection mice were orally gavaged with spiB. (D) Quantification of ileum myenteric plexus neurons at 7 dpi. (E) Serum levels of IL-4 and IL-13 at 7 dpi. (F) Arg1 expression by ileum myenteric plexus macrophages at 7 dpi. (G) Total GI transit time measured 10 days post-spiB infection. (H-L) Epx^ΔIl4;Il13 mice or WT littermates were infected with Sv larvae and 14 days later with spiB. (H) Frequency of duodenum LP eosinophils at 7 dpi. (I) Quantification of ileum myenteric plexus neurons at 7 dpi. (J) Serum levels of IL-4 and IL-13 7 dpi. (K) Arg1 expression by ileum myenteric plexus macrophages at 7 dpi. (L) Total GI transit time measured 10 days post-spiB infection. (M) Experimental design for (N-P). (N-P) C57BL/6J CD45.2 mice received indicated numbers of eosinophils isolated from duodenum or ileum LP of CD45.1 mice infected with Sv 7 days earlier. Recipient mice were subsequently infected with spiB. (N) Frequency of donor CD45.1⁺ duodenum eosinophils found in LP of recipient mice at 7 dpi. Quantification of ileum myenteric plexus neurons in mice receiving duodenum (O) or ileum (P) eosinophils 7 days post-spiB infection. (R) C57BL/6J mice were infected with Sv. Expression of IL-4 was measured in duodenum and ileum eosinophils 7 days post-infection. Dashed lines indicate the range of day 7 enteric neuron numbers defined by mean ± SEM of a set of control non-infected C57BL/6J mice (n=25). Data is from one representative out of 2 independent experiments (A-H and J-R) or 2 pooled independent experiments with 3–5 mice per condition (I). See also Figure S4.

Figure 7.. Long-term neuroprotection is maintained by modulation of hematopoietic progenitors.

(A-E) C57BL/6J mice were infected with Sv. Frequencies of indicated bone marrow progenitor cell subsets 1, 2 and 8 weeks post-Sv infection. (F) Experimental design for (G and H). (G and H) Lethally irradiated CD45.1⁺ host mice received 1000 LT-HSCs isolated from donor CD45.2⁺ mice s.c. injected with water or infected with Sv 14 days earlier. (G and H) Frequency of CD45.2⁺ duodenum and ileum LP eosinophils 4 weeks post-HSC transfer. (I) Experimental design for (J and K). Sublethally irradiated CD45.2⁺ host mice received 1×10⁶ lineage-negative bone marrow cells isolated from donor CD45.1⁺ mice s.c. injected with water or infected with Sv 14 days earlier. After 12 weeks, host mice were infected with spiB. (J) Quantification of ileum myenteric plexus neurons at 7 dpi. (K) Arg1 expression by ileum myenteric plexus macrophages at 10 dpi. (L) Experimental design for (M-O). (M-O) CD45.2⁺ host mice were s.c. injected with water or Sv larvae and 14 days later lethally irradiated and i.v. injected with 1×10⁶ lineage-negative bone marrow cells isolated from donor CD45.1⁺ mice infected with Sv 14 days earlier. After 12 weeks, host mice were challenged with spiB. (M) Quantification of ileum myenteric plexus neurons at 10 dpi. (N) Arg1 expression by ileum myenteric plexus macrophages at 10 dpi. (O) Frequency of CD45.1⁺ and CD45.2⁺ ileum myenteric plexus macrophages in host mice. Data is from one representative out of 2 independent experiments (G-O) or 2 pooled independent experiments with 3–5 mice per condition (B-E). See also Figure S5.