The aryl hydrocarbon receptor regulates lipid mediator production in alveolar macrophages

Allergic inflammation of the airways such as allergic asthma is a major health problem with growing incidence world-wide. One cardinal feature in severe type 2-dominated airway inflammation is the release of lipid mediators of the eicosanoid family that can either promote or dampen allergic inflammation. Macrophages are key producers of prostaglandins and leukotrienes which play diverse roles in allergic airway inflammation and thus require tight control. Using RNA- and ATAC-sequencing, liquid chromatography coupled to mass spectrometry (LC-MS/MS), enzyme immunoassays (EIA), gene expression analysis and in vivo models, we show that the aryl hydrocarbon receptor (AhR) contributes to this control via transcriptional regulation of lipid mediator synthesis enzymes in bone marrow-derived as well as in primary alveolar macrophages. In the absence or inhibition of AhR activity, multiple genes of both the prostaglandin and the leukotriene pathway were downregulated, resulting in lower synthesis of prostanoids, such as prostaglandin E2 (PGE2), and cysteinyl leukotrienes, e.g., Leukotriene C4 (LTC4). These AhR-dependent genes include PTGS1 encoding for the enzyme cyclooxygenase 1 (COX1) and ALOX5 encoding for the arachidonate 5-lipoxygenase (5-LO) both of which major upstream regulators of the prostanoid and leukotriene pathway, respectively. This regulation is independent of the activation stimulus and partially also detectable in unstimulated macrophages suggesting an important role of basal AhR activity for eicosanoid production in steady state macrophages. Lastly, we demonstrate that AhR deficiency in hematopoietic but not epithelial cells aggravates house dust mite induced allergic airway inflammation. These results suggest an essential role for AhR-dependent eicosanoid regulation in macrophages during homeostasis and inflammation

Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing

Concerted Activity of IgG1 Antibodies and IL-4/IL-25-Dependent Effector Cells Trap Helminth Larvae in the Tissues following Vaccination with Defined Secreted Antigens, Providing Sterile Immunity to Challenge Infection

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations

Antibody and Fcrg deficient mice display increased intestinal lesions and reduced accumulation of myofibroblasts.

<p>Mice were challenge-infected with 200 infective <i>Hpb</i> larvae (scheme of infection and antihelminthic regime depicted in (A)), small intestines were harvested at day 10, 14 or 21 p.i.; tissue sections were stained by hematoxillin and eosin (H&E) or immunohistochemistry (IHC) and wide field microscopy images were analyzed using ImageJ. (B) Representative pictures of the largest H&E-stained cross sections of lesions at day 10 p.i. in C57BL/6 or Aid^-/- mice; (C) Quantification of lesion area of largest cross sections (day 10 p.i.) for C57BL/6 or Aid^-/- mice; (D) Representative pictures of the largest H&E-stained cross sections of lesions from C57BL/6, Aid^-/- or Fcrg^-/- mice at day 14 p.i.; (E) Quantification of lesion area of largest cross sections (day 14 or 21 p.i.) for C57BL/6, Aid^-/- or Fcrg^-/- mice; (F) Representative images of IHC staining for aSMA in largest cross sections of lesions in tissues from C57BL/6, Aid^-/- or Fcrg^-/- mice; arrows indicate aSMA⁺ areas with potentially contractile morphology. (G) Quantification of aSMA-stained area in lesions of C57BL/6, Aid^-/- or Fcrg^-/- mice; (H) Quantification of lesion area of largest cross sections (day 14 p.i.) for C57BL/6 or Aid^-/- mice treated with immune serum from secondary infected (IS) or naïve (NS) C57BL/6 mice; (I) Representative pictures of the largest H&E-stained cross sections (top) or IHC staining for aSMA (bottom) of lesions at day 14 p.i. in C57BL/6 or Aid^-/- mice treated with immune serum from secondary infected (IS) or naïve (NS) C57BL/6 mice; (J) Quantification of aSMA-stained area in lesions of C57BL/6, IS, NS or untreated (ctr) Aid^-/- mice; All data are pooled from 2–3 independent experiments with 3–6 mice per group and presented as mean + SEM; Scale bars 200 μm.</p

Inhibition of CXCR2 signaling leads to delayed MF accumulation and increased lesion size without affecting granulocyte recruitment in vivo.

<p>C57BL/6 mice were treated or not with the CXCR2 antagonist SB265610 (3 mg/kg per oral gavage, once daily) during challenge infection with <i>Hpb</i>; small intestines were harvested for parasitology and histology at day 14 p.i. (A) Lesions were counted in small intestines of untreated (control) and SB265610 treated mice; (B) Adult worms were counted in opened small intestines of untreated (control) and SB265610 treated mice; (C) The area of the largest lesion cross sections was quantified in H&E-stained tissue sections from untreated (control) and SB265610 treated mice; (D) Representative H&E-stained cross sections of intestinal lesions in untreated (control) and SB265610 treated mice; Scale bars 200 μm. (E) Representative IHC-staining for aSMA in lesions of untreated (control) and SB265610 treated mice; Scale bars 200 μm. (F) Quantification of aSMA-stained area in lesions of untreated (control) and SB265610 treated mice was performed using ImageJ; (G) Small intestinal tissue sections from challenge-infected untreated C57BL/6 (control) and SB265610 treated C57BL/6 mice or Aid^-/- or Fcrg^-/- mice were IF-stained for the neutrophil marker MPO (upper panel, green) or the eosinophil marker 12/15-lipoxygenase (12/15LO) (lower panel, green), followed by counterstaining with DAPI (blue); Scale bars 50 μm. (H) Total Fluorescence intensity of MPO was quantified using CellProfiler; (I) Total Fluorescence intensity of 12/15LO was quantified using CellProfiler; All data are pooled from at least 2 independent experiments with 3–6 mice per group and presented as mean + SEM.</p

Conditioned media from <i>A</i>. <i>suum</i>-immune serum-activated MΦ promote in vitro scratch wound closure by human MF in a CXCR2 dependent fashion.

<p>(A) Human MDM from 3 healthy blood donors (1–3) were co-cultured with <i>A</i>. <i>suum</i> larvae in the absence or presence of IS from challenge-<i>A</i>.<i>suum</i> infected pigs and time-lapse movies were recorded (2 frames/ s, 1 min); upper panel: representative snap shots at T = 0; lower panel: representative temporal color code images (120 frames, 1 min)—colors represent different time points; Scale bars 100 μm; (B) Adherent MDM per <i>A</i>. <i>suum</i> larva were counted in time-lapse movies; (C) CXCL3 in culture supernatants from human MDM cultured with or without immune sera from <i>Ascaris</i>-infected pigs (IS), <i>Ascaris suum</i> larvae (<i>A</i>.<i>s</i>.) or both (<i>A</i>.<i>s</i>. IS) quantified by ELISA; (D) Scratch wound closure by human MF cultured in the presence of conditioned media from MDM (MDM sup) stimulated with <i>A</i>.<i>s</i>. IS +/- the CXCR2 antagonist SB265610; All data are pooled from at least 3 independent experiments with n = 3 co-cultures in each and presented as mean +/-SEM.</p

Helminths and antibodies trigger CXCL2 release by MΦ or MF via antibody-Fcrg- or Fcrg-chain/ dectin2 signaling, respectively and MF respond to MΦ-produced CXCR2 ligands in vitro.

<p>Bone marrow derived MΦ (BMM) or primary small intestinal MF from were co-cultured with <i>Hpb</i> larvae (L3) for 24h with our without immune sera (IS) from challenge <i>Hpb</i> infected mice; (A) CXCL2 in BMM culture supernatants (C57BL/6 or Fcrg^-/-) cultured without (ctr) or with IS, L3 or both; (B) top: CXCL2 staining in control or L3IS-co-cultured BMM, bottom: IgG (red)/ CXCL2 (green) overlay; (C) Scratch wound closure by MF after addition of supernatants from BMM, cultured without (ctr) or with L3 and IS +/- CXCR2 antagonist SB265610. (D) <i>Cxcl2</i> mRNA induction in MF after IS, L3 or L3 and IS-stimulation relative to unstimulated MF; (E) CXCL2 in culture supernatants from MF, cultured without (ctr) or with L3 or L3 and IS; (F) Overlays of CXCL2 (green) and IgG (red) for unstimulated (control) MF and L3IS-co-cultured C57BL/6 or Fcrg^-/- MF; (G) <i>Cxcl2</i> mRNA induction in MF after L3 co-culture; (H) CXCL2 in MF supernatants after culture without (ctr) or with L3 or L3IS;(I) <i>Cxcl3</i> mRNA induction in MF after culture in the absence or presence of L3 or L3IS; (J) CXCL3 in unstimulated (control) MF or L3-co-cultured MF; (K) <i>Cxcl3</i> mRNA induction in MF after L3-co-culture; All data are pooled from at least 2 independent experiments with cells from 3–4 mice per group and presented as mean + SEM.</p