M2-like, dermal macrophages are maintained via IL-4/CCL24-mediated cooperative interaction with eosinophils in cutaneous leishmaniasis.

Eosinophil IL-4 regulates CCL24 expression and proliferation of peritoneal macrophages during experimental Ascaris infection To evaluate the significance of IL-4-CCL24 mediated interaction between TRMs and eosinophils in other tissue pathologies, we used Ascaris suum , a helminth infection model in the peritoneum which promotes a local eosinophilia( 29 ). Peritoneal fluid from naïve animals already contained a significant amount of CCL24 (300–500 pg/ml), an underestimate considering the fluid was diluted in PBS used to harvest the peritoneal cells ( Fig. 7A and C ). A single intraperitoneal injection of live Ascaris eggs induced a large amount of CCL24 (1–3 ng/ml) that was significantly reduced in anti-IL-4 treated mice ( Fig. 7A ) and in eoCre il4/13 f/f mice lacking IL-4 production from eosinophils ( Fig. 7C ). As expected, marked accumulation of peritoneal eosinophils accompanied the increased CCL24 production after Ascaris infection that was ablated by anti-IL-4 treatment ( Fig. 7B ). The number of peritoneal macrophages was also decreased following IL-4 neutralization, a result consistent with the IL-4 driven, local proliferation of peritoneal macrophages in Brugia malayi , another tissue-invasive nematode( 13 ). Moreover, both CCL24 neutralization as well as selective deficiency of IL-4 from eosinophils strongly reduced peritoneal eosinophil numbers ( Fig. 7D ). Importantly, both CCL24 neutralized WT and eoCre il4/13 f/f mice, CCL24 neutralized or not, had a significantly decreased number of peritoneal TRMs compared to WT mice. While fewer numbers of macrophages were recovered when comparing day 3 Ascaris-infected mice to naïve mice, their adhesion to the parasites and the “macrophage disappearance reaction” that is known to occur after acute inflammation, may have made them more difficult to be recovered in the peritoneal lavage( 30 , 31 ). However, increased proliferation of the peritoneal macrophages was confirmed by both Ki67 staining and BrdU incorporation at 72 hours p.i., which was abrogated in the eoCre il4/13 f/f mice ( Fig. 7E ). To visualize the association of peritoneal TRMs and eosinophils during Ascaris infection, we performed ex vivo live imaging of peritoneal cells harvested from infected mice with or without CCL24 neutralization ( Fig. 7F ; Mov. S13 and S14). We observed clusters of closely interacting cells containing F4/80 + peritoneal macrophages and eosinophils, which showed minimal motility. Of note, cellular material from eosinophils seemed to be directly transferred to or taken up by peritoneal macrophages during their close interaction ( Fig. 7F , inset 1 and 2; Mov. S15 and S16). In contrast, no cluster of cells were formed following CCL24 neutralization, and eosinophils showed increased migration without interaction with peritoneal macrophages ( Fig. 7F and G ). To determine whether peritoneal TRMs, as well as TRMs from other tissues, produce CCL24 in response to IL-4/10, we sorted TRMs from various tissues and stimulated them with IL-4 and/or IL-10 ( Fig. 7H ). Resident macrophages recovered from the peritoneum, lung, liver and adipose tissue all produced a large amount of CCL24 in response to IL-4/10 simulation. IL-4 alone was sufficient to induce CCL24 expression in large peritoneal macrophages and Kupffer cells. In contrast, red pulp macrophages from spleen failed to produce CCL24 under any condition tested. Finally, we compared CCL24 mRNA expression from gene expression profiles of various hematopoietic populations compiled in the ImmGen RNA-Sequencing database( 32 ). Ccl24 expression is restricted to TRMs, including peritoneal and adipose TRMs ( Fig. 7I ).

Materials and Methods Study Design

The goal of these studies was to demonstrate how dermal TRMs maintain the population size and M2-like characteristics during classical T H 1 immune response against L. major infection. We compared the population size and transcriptional profiles of dermal TRMs between WT and eoCre IL4/13 f/f mice. Animals were randomly assigned to different experimental groups. The sample size and reproducibility of experiments are specified in each figure legend. Sample measurements were blinded during experiments or analysis.

Mice

C57BL/6 mice were purchased from Taconic Farms. C57BL/6 CD45.1 + or CD45.2 + rag1 −/− mice, C57BL/6 il4 −/− mice, and C57BL/6 Lyz2 GFP mice were obtained through a supply contract between the National Institute of Allergy and Infectious Diseases (NIAID) and Taconic Farms. ROSA26-LSL-tdTomato mice (also known as Ai14) mice and Actb-DsRed.T3 were purchased from The Jackson Laboratory. Dr. Helene Rosenburg (NIAID) kindly provided C57BL/6 il5 −/− mice. eoCre mice, originally developed by Doyle et al.( 45 ), were obtained from Mayo Clinic, Scottsdale, AZ and crossed with ROSA26-LSL-tdTomato mice to generate eosinophil-specific fluorescent reporter animals( 27 ). eoCre il4/13 f/f mice with selectively deficiency of IL-4/13 in eosinophils( 25 , 26 ), were also obtained from Mayo Clinic, Scottsdale, AZ. All of the mice used in these studies were female, 6–8 weeks old, and were bred and/or maintained in the NIAID animal care facility under specific pathogen-free conditions. They were used under a study protocol approved by the NIAID Animal Care and Use Committee (protocol number LPD 68E). All aspects of the use of animals in this research were monitored for compliance with The Animal Welfare Act, the PHS Policy, the U.S.

Government Principles for the Utilization and Care of Vertebrate Animals

Used in Testing, Research, and Training, and the NIH Guide for the Care and Use of Laboratory Animals. In vivo injections Manocept-Alexa488 or -Cy5 (Navidea Biopharmaceuticals) is a fluorescently labeled derivative of FDA-approved 99MTC-Tilmanocept targeting MR. Naïve mice were injected with 25μg Manocept intravenously with 20μg anti-CD31 Abs (Clone#390, Invitrogen) in total volume not to exceed 100uL per mouse during IVM. For eosinophil depletion, animals were i.p. injected 15μg of SiglecF Abs (Clone# 238047, R&D systems) at day2 p.i. and every two days. In vivo neutralization of CCL24 was carried out by i.p. administration of 100μg of monoclonal CCL24 Abs (Clone# 106521, R&D systems) every day for 3 days beginning one day prior to L. major or Ascaris infection. Leishmania major and Ascaris suum infection The L. major Seidman strain (MHOM/SN/74/SD) (LmSd) was maintained as follows: promastigotes were grown at 26°C in medium 199 supplemented with 20% heat-inactivated FCS (Gemini Bio-Products), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 40 mM Hepes, 0.1 mM adenine (in 50 mM Hepes), 5 mg/mL hemin (in 50% triethanolamine), and 1 mg/mL 6-biotin (M199/S). Parasites expressing a red fluorescent protein (LmSd-RFP) were grown using the identical culture medium supplemented with 50 μg/mL of Geneticin (G418, Gibco, Woodland, CA). Infective-stage, metacyclic promastigotes were isolated from stationary cultures (5–6 days) by density gradient centrifugation, as described previously( 46 ). Mice were then inoculated with metacyclic promastigotes in the ear dermis or footpad by intradermal or subcutaneous injection respectively in a volume of 10μL. Lesion development was monitored weekly by measuring the diameter of the ear nodule with a direct-reading Vernier caliper (Thomas Scientific). Lesion pathology was also evaluated and scored as follows: 0 = no ulceration, 1 = ulcer, 2 = half ear eroded, 3 = ear completely eroded. For intraperitoneal inoculation of Ascaris suum , live eggs were isolated from adult female uteri through gentle mechanical maceration, purified by straining, and cultured to embryonation in 0.2 M H 2 SO 4 for 100 days, which corresponds to the peak of larvae infectivity as described( 47 ). The fully embryonated eggs (containing the L3 infective stages) were used for experimental infections. Mice were inoculated through the intraperitoneal route with 10,000 fully embryonated eggs to induce a local eosinophilia, as previously demonstrated( 29 ). Three days post-injection, the peritoneal cavity cells and lavage were collected. Processing of ear tissues and evaluation of L. major parasite burden Ear tissue was prepared as previously described( 48 ). Briefly, the two sheets of infected ear dermis were separated, deposited in DMEM containing 0.2 mg/mL Liberase TL purified enzyme blend (Roche Diagnostics Corp.), and incubated for 1.5 h at 37°C. Digested tissue was processed in a tissue homogenizer for 3.5 minutes (Medimachine; Becton Dickinson) and filtered through a 70 μm cell strainer (Falcon Products) to obtain single cell suspension. Parasite titrations were performed as previously described( 49 ). Briefly, tissue homogenates were serially diluted in 96-well flat-bottom microtiter plates containing 100 μL M199/S. The number of viable parasites in each ear was determined from the highest dilution at which promastigotes could be grown out after 7–10 days of incubation at 26°C. Isolation of mouse peritoneal cells Ascaris -infected mice were i.p. injected with 1ml of PBS. After injection, the peritoneum was gently massged to dislodge any attached cells into the PBS solution. Cell suspension in PBS were collected with 25g needle. After centrifugation, the supernatant was collected for assays, and cells from the first wash were combined with the second lavage with 3ml PBS.

Detection of CCL24 levels by ELISA

CCL24 levels in the peritoneal lavage of Ascaris -inoculated mice were quantified by ELISA kits purchased from R&D Systems, Inc. (Minneapolis, MN, USA). All assays were performed according to the manufacturers’ protocols.

Immunolabeling and flow cytometry analysis

Detailed methods for the surface and intracellular cytokine staining of ear dermal cells for flow cytometry analysis are described in the Supplemental Materials and Methods . BMDM and BM-derived eosinophil generation, infection, and in vitro assays Isolated femurs and tibia were flushed with PBS, and for BMDM differentiation precursor cells were cultured in complete RPMI supplemented with 30% L929 cell-conditioned medium. The media was changed every 2 days thereafter. After 7d in culture, mature BMDMs were harvested by washing with cold PBS, incubating on ice for 15 minutes, and pipetting extensively. BMDMs were plated at 5 × 10 5 /well onto 24 well plates in AIM V serum-free media (Thermofisher Scientific) with 10ng/ml IL4, IL10, or IL13 with or without purified metacyclic promastigotes. After 3d culture, supernatants were harvested and assayed with Quantibody mouse chemokine array Q1 (Raybiotech). For eosinophil differentiation, precursor cells were cultured in IMDM (Iscove’s Dulbecco’s Media) containing Glutamax (Life Technologies), 10% FCS (HyClone), 2mM L-glutamine (Life Technologies), 50μM β-mercaptoethanol (Sigma-Aldrich), Penicillin/Streptomycin (Life Technologies), and 50ng/ml SCF (Peprotech)( 50 ). On day 4, the media was replaced with fresh complete media containing 10ng/ml murine IL-5 (Peprotech). Media with IL-5 was changed every 2 days thereafter until harvesting fully differentiated eosinophils on day 14.

Eosinophil trans-well migration assay

BMDMs were plated 1 × 10 5 /well to lower wells of HTS Transwell-96 well plates (Corning) and stimulated with 10 ng/ml IL-4, IL-10, or IL-13 in the presence of anti-CCL24 Ab or isotype control (R&D systems). After 72 hours, 2 × 10 5 BM-derived eosinophils were added to the upper wells, and then incubated for 90 min at 37°C. Eosinophil migration was normalized to the respective assays having only IL4, IL10, or IL13 cytokines without BMDMs to exclude chemotactic effect of cytokines. Confocal microscopy, live cell imaging and intravital microscopy Detailed methods for immunostaining and confocal microscopy of cryostat sections of infected ears, for live cell imaging of BMDM co-cultured with eosinophils in vitro , for imaging peritoneal cells ex vivo , and for non-invasive intravital imaging of the mouse ear, are described in the Supplemental Materials and Methods . BM chimeras Recipient mice were irradiated with a single dose of 1,250 rads and reconstituted with donor bone marrow. The harvested BM cells were injected intravenously at a dose of 2.5 × 10 6 cells/mouse in 100 µl PBS. Animals were maintained on trimethoprim-sulfamethoxazole (Hi-Tech Pharmacal) antibiotic water for up to 5 weeks after irradiation. Hematopoietic reconstitution of all animals was verified by flow cytometry staining of congenic markers (CD45.1 and 45.2).

RNA sequencing and data analysis

Single cell suspension and antibody staining were performed as described above except for addition of 100 unit/ml SUPERase•InTM RNase Inhibitor (ThermoFisher) to all of the tissue-digestion/staining buffers to inhibit the strong ribonuclease activity in murine skin. One hundred dermal TRMs were sorted directly into lysis buffer included in SMART-Seq V4 Ultra Low Input RNA Kit for Sequencing (Takara), and cDNA was generated according to the commercial protocol. Detailed methods for library preparation, sequencing, and bioinformatic analysis are described in the Supplemental Materials and Methods .

Statistical analyses

The differences in values obtained for two different groups were determined using non-parametric Mann-Whitney test. For comparisons of multiple groups, one-way analysis of variance (ANOVA) followed by Dunn’s post-test was used. Analyses were performed using Prism 8.0 software (GraphPad).

Supplementary Material Table 1 Table S1. List of differentially expressed genes (DEGs). Table 2 Table S2. List of enriched GO categories. Table 3 Movie 1 Movie S1. 3D rotational confocal imaging of inoculation site and the area proximal to the inoculation site. Movie 2 Movie S2. In vitro live imaging of eosinophils and IL-4/10 stimulated BMDMs with or without CCL24 neutralization. Supplemental material Supplemental Materials and Methods Fig. S1. Gating strategy for eosinophils, PMNs, Ly6C hi monocytes, Ly6C int /Ly6C lo moDCs, and dermal TRMs. Fig. S2. Basophils and ILC2s produce IL-4. Fig. S3. Dermal TRMs are the main producers of Ccl24 among dermal myeloid subsets. Fig. S4. Eosinophils progressively infiltrate the L. major inoculation site. Fig. S5. Immunofluorescence staining and confocal microscopy on vertical ear sections of infected eoCre il4/13 f/f and WT mice. Movie 3 Movie S3. Intravital imaging of perivascular dermal TRMs. Movie 4 Movie S4. Intravital imaging of the active surface movements of dermal TRMs. Movie 5 Movie S5. Intravital imaging of eosinophils with dermal TRMs in steady state. Movie 6 Movie S6. Intravital imaging of eosinophils and dermal TRMs at 1h p.i. with or without CCL24 neutralization. Movie 7 Movie S7. Intravital imaging of eosinophils and dermal TRMs at 3d p.i. with or without CCL24 neutralization. Movie 8 Movie S8.

Ex vivo live imaging of eosinophils and peritoneal

TRMs with or without CCL24 neutralization. Movie 9 Movie S9. Ex vivo live imaging of eosinophils transferring cellular materials to peritoneal TRMs.