Skin punch biopsies were collected as described in the section “CGRP release assay from skin explants” from the flank area injected with vehicle or bacteria. Samples were collected under sterile conditions, rapidly transferred to sterile 24-well cell culture plates (Genesee Scientific, Cat# 25-107) containing 1 mL of DMEM (Thermo Fisher, Cat# 11995073) at 32°C and immediately used for experiments.

For the experiments involving in vitro CGRP release assays by neurons, or co-incubation of DRG neurons with neutrophils, DRG neurons were first incubated for one week in laminin (20 μg/mL, Thermo Fisher, Cat# 23017015)-coated sterile flat bottom 96-wells plates (Thermo Fisher, Cat# 08-772-53) containing 200 μL of neurobasal-A medium (Thermo Fisher, Cat# 21103049), B-27 supplement (Thermo Fisher, Cat# 1704044), penicillin/streptomycin (100 units/mL and 100 μg/mL, Thermo Fisher, Cat# 15140122), L-glutamine (2 mM, Thermo Fisher, Cat# 25030081), mouse NGF (50 ng/mL, Thermo Fisher, Cat# 50385MNAC50), and cytosine arabinoside (10 μM, Sigma, Cat# C6645). Half of the medium was replaced with fresh media every two days. At day 7, neurons were stimulated for CGRP assays or co-cultured with neutrophils for experiments as described in Neuronal stimulation and CGRP release and in Neutrophil isolation and killing assays sections.

All procedures related to bacterial strains and infectious disease work were approved by the Committee on Microbiological Safety (COMS) at Harvard medical school and were conducted under Biosafety Level 2 protocols and guidelines. All Streptococcus pyogenes strains used in this study are listed in the Key Resources Table . S. pyogenes 854 M-type 1 was originally isolated from a patient with a retroperitoneal abscess (). S. pyogenes 950771 M3 serotype strain is a clinical isolate obtained from a patient with necrotizing fasciitis and sepsis (). These founder bacterial strains were mutated to generate isogenic mutant strains (see Method Details ). Bacteria were grown on Tryptic Soy Agar (TSA) plates supplemented with 5% Sheep Blood (BD Biosciences, Cat# 221239), or grown in liquid culture in Todd-Hewitt Broth (Sigma, Cat# T1438) supplemented with 0.2% yeast extract (Sigma, Cat# Y1625) (THY broth) at 37°C with 5% CO. When required, THY broth was supplemented with spectinomycin (50 μg/mL, Sigma, Cat# S4014). For storage, bacterial glycerol frozen stocks (20% glycerol, Sigma, Cat# G5516) of S. pyogenes strains were prepared and kept at −80°C until use.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School and were conducted in accordance with National Institutes of Health (NIH) animal research guidelines. C57BL6/J, B6(Cg)-Rag2 tm1.1Cgn /J, B6N.129S2-Casp1 tm1Flv /J, B6.129P2(SJL)-Myd88 tm1.1Defr /J, B6.129-Trpv1 tm1(cre)Bbm /J, and B6.129P2-Gt(ROSA)26Sor tm1(DTA)Lky /J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were bred and housed in individually ventilated micro isolator cages within a full barrier, specific pathogen-free animal facility at Harvard Medical School under a 12 h light/dark cycle with ad libitum access to food and water. B6.129-Trpv1 tm1(cre)Bbm /J heterozygous (+/−) mice were bred with B6.129P2-Gt(ROSA)26Sor tm1(DTA)Lky /J homozygous (+/+) mice to generate nociceptor-ablated Trpv1-Cre/Dta (Trpv1-Cre +/− /Dta +/− ) mice and control littermates (Trpv1-Cre −/− /Dta +/− ). Both male and female age-matched mice from 6 to 14 weeks of age were used for all experiments in this study. Male and female mice were similarly susceptible to Streptococcus pyogenes infection. Individual animal health status was routinely monitored by Harvard Center for Comparative Medicine veterinary staff. Additionally, one cage containing two sentinel animals was maintained on each rack. These sentinel cages were supplied with pooled samples of soiled bedding from regular colony animals at every cage change. All cage changes were performed in Class II biosafety change stations. Animal sentinels were tested quarterly for pinworms, fur mites, Sendai virus, Pneumonia virus of mice, Mouse hepatitis virus, GD-7 virus, Minute virus of mice, Mycoplasma pulmonis, Mouse parvovirus, Epizootic diarrhea of infant mice, Reo-3 virus, and annually for ectromelia virus and lymphocytic choriomeningitis virus. Only healthy animals were used for experiments. Euthanasia was performed by CO 2 inhalation.

Blood samples were collected by a trained phlebotomist from three healthy adult volunteers of either sex at Boston Children’s Hospital with approval from the hospital’s Institutional Review Board (protocol X04-01-008). Written informed consent was obtained from all volunteers. No further demographic data was collected from the volunteers. Blood collected was from a single donor per experiment and was not pooled.

Method Details

Generation of isogenic mutant S. pyogenes strains Love et al., 2012 Love J.F.

Tran-Winkler H.J.

Wessels M.R. Vitamin D and the human antimicrobial peptide LL-37 enhance group a streptococcus resistance to killing by human cells. Bricker et al., 2002 Bricker A.L.

Cywes C.

Ashbaugh C.D.

Wessels M.R. NAD+-glycohydrolase acts as an intracellular toxin to enhance the extracellular survival of group A streptococci. TCTAGA GGTAACCTTGTTACTGCTAATGC-3′ and 5′-CCC GGATCC CAGTGACAGAGTCAATGATGG-3′ (441 bp product) and 5′-CCC GAATTC GCGGGTGTCAATAACAGAACTG-3′ and 5′-CCC GGTACC CCATATGGGCTCAGGGTTGATC-3′ (367 bp product), respectively. All PCR reactions were performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs). The products were directionally cloned into the temperature-sensitive shuttle vector pJRS233 (provided by June Scott) ( Perez-Casal et al., 1993 Perez-Casal J.

Price J.A.

Maguin E.

Scott J.R. An M protein with a single C repeat prevents phagocytosis of Streptococcus pyogenes: use of a temperature-sensitive shuttle vector to deliver homologous sequences to the chromosome of S. pyogenes. The slo-negative derivatives of the S. pyogenes 854 and 950771 were generated by allelic exchange as described previously (). Genomic DNA for use as a PCR template was prepared from an overnight culture of S. pyogenes as follows: Cells from a 10 mL overnight culture were harvested by centrifugation at 10,000 rpm and resuspended in 1 mL of STE buffer (100 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 8.0) containing mutanolysin (50 μg, Sigma). This suspension was incubated at 37°C with agitation for 2 h at which point DNase-free RNase (10 μg; Sigma) and N-Lauroylsarcosine (0.5% (v/v), Sigma) were added. After a further 10 min incubation at 37°C, pronase was added (10 mg, Sigma) and the mixture was incubated at 37°C for a further 10 min. S. pyogenes genomic DNA was precipitated by mixing with an equal volume of phenol:chloroform (1:1), followed by centrifugation at 12,000 rpm. The pellet was washed with ice-cold ethanol and then washed and dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). For PCR, 1 μL of genomic DNA template in a 50 μL PCR reaction mixture was used. PCR products corresponding to the 5′ and 3′ ends of slo were produced using the primer pairs 5′- CCCGGTAACCTTGTTACTGCTAATGC-3′ and 5′-CCCCAGTGACAGAGTCAATGATGG-3′ (441 bp product) and 5′-CCCGCGGGTGTCAATAACAGAACTG-3′ and 5′-CCCCCATATGGGCTCAGGGTTGATC-3′ (367 bp product), respectively. All PCR reactions were performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs). The products were directionally cloned into the temperature-sensitive shuttle vector pJRS233 (provided by June Scott) () using unique XbaI, BamHI, EcoRI and Asp718 restriction endonuclease sites (underlined) incorporated into the PCR primers. The final construct representing the slo sequence with an 818 bp internal deletion was cloned into pJRS233 at the XbaI and Asp718 sites. The resultant plasmid, pJsloΔ was verified by DNA sequencing (Genewiz) using the primers pJRSseqF (5′-GGGATGTGCTGCAAGGCG-3′) and pJRSseqR (5′-ACGACAGGTTTCCCGACTG-3′), which recognize regions outside the 5′ and 3′ termini of the pJRS233 multiple cloning site. Perez-Casal et al., 1993 Perez-Casal J.

Price J.A.

Maguin E.

Scott J.R. An M protein with a single C repeat prevents phagocytosis of Streptococcus pyogenes: use of a temperature-sensitive shuttle vector to deliver homologous sequences to the chromosome of S. pyogenes. The pJsloΔ plasmid was introduced into competent E. coli DH5α cells (New England Biolabs) by transformation according to the manufacturer’s instructions. This plasmid was purified using the Plasmid midi- or maxi-prep kits (QIAGEN) and transformed into electrocompetent S. pyogenes cells. Plasmid integration followed by allelic exchange was allowed to occur at the permissive temperature (). Mutants were verified by PCR of the slo gene using the first and forth primers listed above and Phusion High-Fidelity PCR Master Mix (New England Biolabs). Sierig et al., 2003 Sierig G.

Cywes C.

Wessels M.R.

Ashbaugh C.D. Cytotoxic effects of streptolysin o and streptolysin s enhance the virulence of poorly encapsulated group a streptococci. GGATCC CACATAGTTATTGATAGAAT-3′ and 5′-TCCAGGAGCAACTTGAGTTG-3′ (5′) and 5′-CAACTCAAGTTGCTCCTGGACAAGGTGGTAGCGGAAGTTA-3′ and 5′-GCG AAGCTT GTAATCCGATAAGGACAAGT-3′ (3′). These fragments have overlapping ends at the 3′ and 5′ ends, respectively to permit a subsequent overlap PCR, using the first and fourth primers listed above. The resultant PCR product harbored an internal 60-bp deletion in sagA. This product was directionally cloned into pJRS233 using the unique BamHI and HindIII restriction endonuclease sites included in the PCR primers (underlined), to generate pJsagAΔ in E. coli strain DH5α. Plasmid pJsagAΔ was purified and used to generate 950771ΔsagA and 854ΔsagA as described above for Δslo. Mutants were identified by their lack of beta hemolysis on blood agar and verified by PCR analysis of the sagA locus. To generate ΔsloΔsagA mutants, pJsagAΔ was introduced to 950771Δslo or 854Δslo by allelic replacement as described above. SLS was inactivated in S. pyogenes 854 and 950771 by deletion of sagA (). DNA fragments corresponding to regions at the 5′ (359 bp) and 3′ (392 bp) termini of sagA were generated using the primer pairs 5′-CGCCACATAGTTATTGATAGAAT-3′ and 5′-TCCAGGAGCAACTTGAGTTG-3′ (5′) and 5′-CAACTCAAGTTGCTCCTGGACAAGGTGGTAGCGGAAGTTA-3′ and 5′-GCGGTAATCCGATAAGGACAAGT-3′ (3′). These fragments have overlapping ends at the 3′ and 5′ ends, respectively to permit a subsequent overlap PCR, using the first and fourth primers listed above. The resultant PCR product harbored an internal 60-bp deletion in sagA. This product was directionally cloned into pJRS233 using the unique BamHI and HindIII restriction endonuclease sites included in the PCR primers (underlined), to generate pJsagAΔ in E. coli strain DH5α. Plasmid pJsagAΔ was purified and used to generate 950771ΔsagA and 854ΔsagA as described above for Δslo. Mutants were identified by their lack of beta hemolysis on blood agar and verified by PCR analysis of the sagA locus. To generate ΔsloΔsagA mutants, pJsagAΔ was introduced to 950771Δslo or 854Δslo by allelic replacement as described above. Nizet et al., 2000 Nizet V.

Beall B.

Bast D.J.

Datta V.

Kilburn L.

Low D.E.

De Azavedo J.C. Genetic locus for streptolysin S production by group A streptococcus. GAATTC GGCCCAAGAACGGAGTGTAT-3′ and 5′-GGA GCATGC TTATTTACCTGGCGTATAACTTCCG-3′. This product was directionally cloned using EcoRI and SphI (underlined in primer sequence) into pDL278, to generate pSagA. Plasmid pSagA was purified and introduced into ΔsagA and ΔsloΔsagA backgrounds by electroporation and maintained by selection using spectinomycin (Sigma). Positive transformants were verified by PCR and their restoration of beta hemolysis on blood agar medium. For complementation studies, the sagA gene including a 780 bp promoter region upstream () was cloned using the primer pair 5′-CCGGGCCCAAGAACGGAGTGTAT-3′ and 5′-GGATTATTTACCTGGCGTATAACTTCCG-3′. This product was directionally cloned using EcoRI and SphI (underlined in primer sequence) into pDL278, to generate pSagA. Plasmid pSagA was purified and introduced into ΔsagA and ΔsloΔsagA backgrounds by electroporation and maintained by selection using spectinomycin (Sigma). Positive transformants were verified by PCR and their restoration of beta hemolysis on blood agar medium.

Streptolysin O activity SLO activity was measured by determination of hemolytic titers of bacterial supernatants of S. pyogenes taken at early stationary phase. Supernatants were filtered through a 0.45 μm membrane, dithiothreitol was added to a final concentration of 6 mM, and the supernatants were incubated at 37°C for 30 min. Sheep erythrocytes were prepared by diluting fresh defibrinated sheep blood (Northeast Lab Services) in PBS and the suspension was added to each bacterial culture sample. After 30 min incubation at 37°C, cell mixtures were centrifuged, and absorbance measured at 550 nm. Hemolytic units correspond to the reciprocal of the dilution of supernatant that yielded 50% lysis, where 100% lysis corresponds to that caused by 1% Triton X-100. Hemolytic activities were also determined after pre-treatment of samples with the SLO inhibitor, cholesterol (cholesterol–methyl-β-cyclodextrin, Sigma) at a concentration of 250 μg/mL (estimated cholesterol concentration, 10 μg/mL).

General experimental design All in vivo experiments were performed in both male and female age-matched littermates. Pain behavioral tests were performed by blinded observers that were unaware of treatment groups and genotypes. Treatment groups of mice were randomized and evenly distributed across both male and female littermates in cages. Treatments were performed by blinded investigators unaware of the contents of syringes or other administration devices. In experiments involving transgenic mice, littermates with different genotypes were cohoused for the duration of experiments. Quantification and analysis of microscopy images were performed by a blinded investigator unaware of groups and genotypes. Animal numbers for pain behavioral analysis and infection outcome measurements were estimated based on pilot studies of S. pyogenes infections in our lab and based on standard numbers used in the field based on publications on pain and bacterial infection work. For pain behavioral tests, we used at least 4 mice per group. For lesion size and pain behavioral experiments, mice that did not survive the entire time-course of analysis were excluded from analysis. For bacterial infections and analysis (demornecrotic lesion size, abscess size, and weight loss following infection), we used at least 5 mice per group. For histology, bacterial load recovery analysis, flow cytometry, ex vivo, and in vitro experiments, we used at least 3 biological replicates per group. All statistical analysis was performed using Graphpad Prism (v. 7.02). For specific numbers of replicates used for each experiment, please see the section Quantification and Statistical Analysis

Bacterial infections S. pyogenes strains were grown overnight on TSA plates supplemented with 5% Sheep Blood (BD Biosciences, Cat# 221239) at 37°C with 5% CO 2 . The next morning, bacterial colonies were picked and inoculated into THY broth (Todd-Hewitt Broth, Sigma, Cat# T1438, with 0.2% yeast extract, Sigma, Cat# Y1625), incubated for 3 h at 37°C without shaking until growth reached mid-exponential phase, and resuspended in fresh medium to A 600nm of 0.6. Bacterial cells were collected by centrifugation at 800g for 15 min, washed once in PBS, and then resuspended in PBS at different estimated concentrations for injection. Before injection of bacteria, mice were lightly anesthetized by inhalation of isoflurane (Patterson Veterinary) 3% in oxygen using a precision vaporizer. For hind paw infections, a single dose of 5x105 – 5x108 cfu of S. pyogenes in 20 μL PBS was administered by intraplantar injection of the right hind paw using a 0.5 cc syringe fitted with a 31-gauge needle (BD Biosciences). For flank infections, 5x106 cfu of S. pyogenes in 50 μL PBS was injected subcutaneously into the flank previously shaved using a hair clipper (Patterson Veterinary). In some cases, hair removal cream (Nair) was also applied for hair removal prior to infection. The bacterial suspension was kept on ice until use, and the inoculum was confirmed by quantitative culture of an aliquot of the final suspension prior to injection. Injections of bacteria or vehicle were performed by an investigator blinded to the content of syringes. Syringes were previously assigned to specific animals by an investigator aware of the groups in order to distribute groups across multiple cages.

Bacterial load recovery analysis Mice were euthanized by CO 2 inhalation and rapidly used for tissue dissection. During necropsy of mice, total hind paw tissues including epidermis, dermis, and subcutaneous tissue to the tendons, or flank tissue encompassing the injection site including epidermis, dermis, and subcutaneous tissue, or spleens were dissected and weighed. Tissues were then transferred to 2 mL eppendorf tubes containing 5 mm stainless steel beads (QIAGEN, Cat# 69989) and 1 mL of ice-cold sterile distilled water. Tissues were homogenized using a TissueLyser II (QIAGEN) for 10 min at 30 Hz. To determine bacterial load recovery, serial dilutions were made and plated on TSA plates with 5% Sheep Blood plates (BD Biosciences, Cat# 221239), and colonies were counted after overnight incubation at 37°C with 5% CO 2 .

Pain behavioral tests Spontaneous pain behaviors were evaluated by quantifying the time that mice spent 1) lifting/licking the hind paw and 2) the number of paw flinches that occurred over a 1 h time period immediately after infection. Data was collected in 5-minute intervals. For mechanical and heat hyperalgesia tests, mice were allowed to habituate to the apparatus during 2 h and for three consecutive days before the beginning of measurements. After habituation, baseline measurements were obtained on two consecutive days prior to infection. Pain intensity to mechanical stimulus (mechanical hyperalgesia) was measured using von Frey monofilaments. Briefly, mice were placed on an elevated wire framework and a series of von Frey monofilaments with different pressure intensities (from 0.007 g to 4g) were applied to the plantar surface of the infected hind paw. The threshold of pain was determined as the lowest pressure filament that induced a response (paw withdrawal) in five out of ten applications. To measure pain sensitivity to a heat stimulus (heat hyperalgesia), mice were placed on the temperature-controlled (29°C) glass plate of a Hargreaves apparatus (Model 390G, IITC Life Science). A radiant heat source (active intensity 23%) was used to stimulate the infected paw by gradually increasing the temperature of the plantar surface. The threshold of pain was determined as the latency (in seconds) to evoke a response of paw withdrawal. The mean of three measurements was determined for each animal at each time point. An exposure limit of 40 s was used to prevent tissue damage. Data from mice that did not survive until the end of the tests were not included in the analysis. Pain behavior tests were performed by blinded observers that were unaware of the treatments, groups, and genotypes.

Lesion size measurement One day prior to subcutaneous injection with S. pyogenes, mice were lightly anesthetized by inhalation of isoflurane (Patterson Veterinary) 3% in oxygen using a precision vaporizer, and the flank area was shaved using a hair clipper. Abscess sizes and dermonecrotic skin lesions were measured daily with a digital caliper (VWR International, Cat# 62379-531) for 14 days after injection, and area calculated with the formula A = (π/2)(length)(width). Mice were also anesthetized with isoflurane 3% in oxygen during the measurements. Data from mice that did not survive until the end of the tests were not included in the analysis.

In vivo BoNT/A and BIBN4096 treatments Botulinum neurotoxin A (BoNT/A, List Biological Labs, Cat# 130B) intrathecal or local subcutaneous pre-treatments were used that were able to distinguish the contribution of pain perception from peripheral neuropeptide release. Mice were subsequently infected with M1 S. pyogenes in the footpad for pain behavioral assays or in the flank for dermonecrotic lesion measurements. For intrathecal pre-treatments, BoNT/A (25 pg in 5 μL PBS) or vehicle (5 μL PBS) was injected at the level of L4-L6 segments of spinal cord 24 h before infection with S. pyogenes. For local pre-treatments, different groups of mice received a subcutaneous BoNT/A injection (25 pg in 5 μL PBS) or vehicle (5 μL PBS) in the footpad, or they received BoNT/A (25 pg in 100 μL PBS) or vehicle (100 μL PBS) in the flank skin at the anticipated site of infection 6 days before infection. We also evaluated whether BoNT/A or CGRP antagonist (BIBN4096, Tocris, Cat# 4561) was able to treat S. pyogenes infection. For these experiments, mice were infected in the flank skin with S. pyogenes M1. 2 h after infection, we performed subcutaneous injection of BoNT/A (25 pg in 50 μL PBS) at the site of infection or intraperitoneal injection of CGRP receptor antagonist BIBN4096 (30 mg/kg). In another set of experiments to determine therapeutic potential, BoNT/A or vehicle was administered locally in mice infected with S. pyogenes at day 2 after flank skin infection, and again at day 9 after infection. For this experiment, BoNT/A (25 pg in 100 μL PBS) or vehicle (100 μL PBS) was distributed in 5 applications of 20 uL each around the borders of the lesion at day 2 and at day 9. The injection sites were marked using a blue marker at each day of injection. BoNT/A was administered using a Hamilton syringe (Hamilton Company, Cat# 7636-01) fitted with a 32 gauge needle (Hamilton Company, Cat# 7803-04). S. pyogenes M1 wt was used at the following doses for the experiments described above: 5x106 cfu for flank infections and lesion size measurements, 5x107 cfu for foot pad infections and hyperalgesia studies, and 5x108 cfu for foot pad infections and spontaneous lifting/licking/flinching tests.

Histology Mice were euthanized by CO 2 inhalation and intracardially perfused with 30 mL of ice-cold PBS, followed by 30 mL of PBS/4% paraformaldehyde (PFA, Sigma, Cat# P6148). Infected hind paw and flank lesion samples were dissected, post-fixed for 12 h at 4°C in PBS/4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained using hematoxylin and eosin (H&E) or Brown and Brenn Gram stains by the Harvard Medical School Rodent Histopathology Core. Stained sections were imaged by light microscopy on an Eclipse Ti-S/L100 inverted microscope (Nikon), and images collected by NIS-Elements AR software.

Immunostaining and microscopy For immunofluorescence staining, hind paw skin tissues and dorsal root ganglion (DRG) tissues were dissected from mice previously euthanized by CO 2 inhalation and intracardially perfused with 30 mL of PBS, followed by 30 mL of PBS/4% PFA (Sigma, Cat# P6148). Samples were post-fixed in PBS/4% PFA solution at 4°C for 12 h, cryoprotected in PBS/30% sucrose (Sigma, Cat# S0389) for 3 days at 4°C, embedded in Optimal Cutting Temperature (OCT, Sakura Finetek, Cat# 4583), and stored at −80°C until processing. Cryosections (20 μm for DRG, 40 μm for skin) were cut onto Superfrost Plus slides (Thermo Fisher) before immunostaining. Hind paw skin or DRG sections were stained with mouse or rabbit anti-beta III tubulin (Tuj1, Abcam, 1:500), rabbit anti-CGRP (Sigma, 1:10,000), mouse anti-NF200 (MilliporeSigma, 1:1000), mouse anti-PGP9.5 (Abcam, 1:500), or guinea pig anti-TRPV1 (MilliporeSigma, 1:1000), followed by Alexa 594 donkey anti-mouse IgG (Abcam, 1:500), DyLight 488 donkey anti-rabbit IgG (Abcam, 1:500) or goat anti-guinea pig IgG (Sigma, 1:500). Stained sections were mounted in Vectashield mounting medium (Vector Labs, Cat# H1000), with addition of DAPI (BioLegend, Cat# 422801) for skin samples. Fluorescence imaging was performed using a FV1000 laser-scanning confocal microscope (Olympus). Data were collected using Olympus Fluoview software. Samples were imaged with z stacks of 1 μm steps and 20 μm (for DRG) or 40 μm (for skin) total thickness; maximum projection images were exported for analysis.

Quantification of DRG neurons DRG samples were collected, sectioned, stained, and imaged as described in the section Immunostaining and microscopy. Maximum projection images (3 fields per sample) obtained for each channel were exported for analysis. The number of TRPV1, CGRP, or NF200 positive neurons, and the total number of neurons (βIII-tubulin positive) field were quantified by an investigator blinded for the groups. Percentage of TRPV1, CGRP, or NF200 positive neurons out of the total neurons (βIII-tubulin positive) was determined for each sample as the average of 3 fields.

RTX mediated ablation of nociceptor neurons Riol-Blanco et al., 2014 Riol-Blanco L.

Ordovas-Montanes J.

Perro M.

Naval E.

Thiriot A.

Alvarez D.

Paust S.

Wood J.N.

von Andrian U.H. Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Resiniferatoxin (RTX, Sigma-Aldrich), a potent capsaicin analog, was used to deplete TRPV1-positive nociceptors. Male and female 4-week-old C57BL/6 mice were lightly anesthetized by inhalation of isoflurane (Patterson Veterinary) 3% in oxygen using a precision vaporizer. Three RTX escalating doses (30 μg/kg, 70 μg/kg, 100 μg/kg, diluted in PBS with 1.2% DMSO and 0.06% Tween-80) were subcutaneously injected over the flank of anesthetized mice on three consecutive days, as adapted from established protocols (). Control littermates were injected with vehicle solution on the same days (PBS with 1.2% DMSO and 0.06% Tween-80). Mice were used for infection experiments four weeks after the last injection of RTX. Vehicle and RTX treated mice were housed together before and during the experiments. Efficiency of RTX treatment in depleting TRPV1-positive nociceptors was confirmed by counting the number of TRPV1-positive neurons in the DRG by microscopy. DRG samples were collected, sectioned, and stained as described in the section Immunostaining and microscopy, and quantified as described in the section Quantification of DRG neurons.

Dorsal root ganglia neuron dissection and culture Adult, 7 – 13 wk old male and female mice were euthanized by CO 2 inhalation. Dorsal root ganglia (DRG) were dissected from all segments of the spinal cord and transferred to neurobasal medium (Thermo Fisher) supplemented with B-27 (Thermo Fisher) and penicillin/streptomycin (Thermo Fisher). DRGs were enzymatically dissociated by incubating in 2 mL of HEPES-buffered saline (Sigma) containing collagenase A (1 mg/kg, Sigma) and dispase II (2.4 U/mL, Roche Applied Sciences) for 20 min at 37°C. Supernatant was carefully removed, replaced with 2 mL of fresh collagenase A/dispase II solution and incubated for 20 min at 37°C again. Cells were transferred to a tube containing 10 mL of DMEM/10% FBS (Thermo Fisher), centrifuged for 1 min at 200g at 4°C, and resuspended in 800 μL of DMEM/10% FBS containing DNase I (150U/mL, Thermo Fisher). DRG cells were dissociated with fire-polished glass Pasteur pipettes (VWR International) with decreasing tip diameters to create single-cell suspensions. Cells were resuspended in 2 mL of neurobasal medium (Life Technologies), and then centrifuged (260g, 10 min) after overlaying on a 10% bovine serum albumin (BSA) gradient (diluted in Neurobasal medium from a 30% BSA solution in PBS, Sigma). Supernatant was removed and resulting pellet resuspended in neurobasal medium for plating. For calcium imaging, cells were plated onto 35 mm laminin-coated (Thermo Fisher) cell culture dishes (2,000 cells per dish) in neurobasal-A medium plus 50 ng/mL nerve growth factor (Thermo Fisher); DRG neurons were used for calcium imaging 12 – 24 h after plating. For co-incubation with neutrophils and CGRP release experiments, 5,000 DRG neurons were seeded per well in laminin-coated flat bottom 96-wells plates and incubated with neurobasal-A medium plus 50 ng/mL nerve growth factor (Thermo Fisher) and cytosine arabinoside (10 μM, Sigma) for one week; half of the medium was replaced with fresh media every two days.

Calcium imaging and data analysis Cultured DRG neurons were washed and loaded with 5 μM Fura-2 AM (Thermo Fisher) in Neurobasal-A medium for 30 min at 37°C, then washed twice and imaged in Krebs-Ringer solution (Boston BioProducts). DRG neurons were imaged using an Eclipse Ti-S/L100 inverted microscope (Nikon) and Zyla sCMOS camera (Andor). An ultraviolet light source (Lambda XL lamp, Sutter Instrument) was used for excitation of Fura-2-AM by alternating 340 nm and 380 nm wavelengths. NIS-elements software (Nikon) was used to image, process and analyze 340/380 ratiometric images from neurons. An increase in 340/380 ratio of 10% or more from baseline levels was considered a positive response to a ligand. For calcium imaging experiments, cell size for individual DRG neurons (measured as area in μm2) was determined using NIS-elements software by marking individual cells using the Region of Interest tool in combination with the Automated Measurement tool (Nikon). The percentage of bacteria-responsive cells or bacteria-unresponsive cells from 3 separate neuronal fields/condition was determined and binned into four groups for analysis based on their cell body area (< 149, 150-249, 250-349, and > 350 μm2).

S. pyogenes supernatant for neuronal stimulation S. pyogenes strains were grown overnight on TSA plates with 5% sheep blood (BD Biosciences) at 37°C in 5% CO 2 . Bacterial colonies were picked and inoculated into liquid cultures of THY broth, grown for 3 h at 37°C without shaking until mid-exponential phase, and bacterial density estimated by A 600nm . Bacterial cells were collected by centrifugation, washed, and then resuspended (5x108-5x1010 cfu per mL) in phenol red-free neurobasal-A medium plus 6% BSA (Sigma) and incubated at 37°C for 1 h, centrifuged for 15 min at 800g, and the supernatant filtered with a 20 μm cell strainer. For calcium imaging, DRG neurons were stimulated with 200 μL of the filtrate, representing bacterial supernatant. For CGRP release assay, 50 μL of bacterial supernatant was used.

Neuronal stimulation and CGRP release DRG neurons (5,000 per well) were cultured for one week in Neurobasal-A medium containing 50 ng/mL nerve growth factor and cytosine arabinoside as described in the sections Dorsal root ganglia neuron dissection and culture. One group of neurons was treated with 25 pg (in 200 uL of Neurobasal-A medium) of Botulinum neurotoxin A (BoNT/A) for 24 h prior to neuronal stimulation. The neuronal culture medium was removed from all wells, and 200 μL of fresh neurobasal-A medium was added to the wells. Filtered supernatants from S. pyogenes M1 854 strain or isogenic mutant strains were collected at the day of the test as described in the section S. pyogenes supernatant for neuronal stimulation. Immediately before stimulation, 50 μL of cell culture supernatant was removed and 50 μL of bacterial supernatant or control medium (neurobasal-A medium + 6% BSA) was added to the wells. Cells were incubated for 30 min at 37°C and with 5% of CO 2 and then 50 μL of supernatant were collected to determine CGRP concentration. A CGRP Enzyme Linked Immunosorbent kit (Cayman Chemical) was used to quantify CGRP according to manufacturer`s instructions.

Neutrophil isolation and killing assays Following euthanasia, femurs and tibias were dissected from mice. Bone marrow cells were flushed out using PBS/1 mM EDTA (Sigma) in a syringe and a 21 gauge needle. Cells were then strained through a 100 μm cell strainer, centrifuged for 5 min at 300g, resuspended in 3 mL of red blood cell lysis buffer (eBioscience) and incubated for 15 min at room temperature. PBS (22 mL) was added and the cells were centrifuged for 5 min at 300g. Supernatant was removed, and the cells were resuspended in neurobasal-A medium (Thermo Fisher) with 10% fetal bovine serum (FBS) at a maximum concentration of 1x108 cells/mL. Neutrophils were isolated using an immune magnetic negative selection kit according to manufacturer’s instructions (EasySep mouse neutrophil enrichment kit and EasySep Magnet, StemCell). Half of the final neutrophil suspension solution was saved to collect the supernatant. This supernatant was added to the conditions where neutrophils were absent (control conditions) instead of fresh media to control for effects of used cell media on bacterial growth. For opsonophagocytic killing assays, mouse neutrophils were used immediately after isolation. S. pyogenes (5x103 cfu) M1 wt strain was mixed with mouse neutrophils (5x105 cells per well) in 200 μL of neurobasal-A medium containing 10% of fresh mouse serum. As described before, neutrophil filtered supernatant was added to the control conditions (without neutrophils). CGRP (1 μM, GenScript) or the antagonists CGRP 8-37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes. For neuron-neutrophil co-incubation experiments, neutrophils and bacteria were added to the plates containing 5x103 DRG neurons and incubated under the same conditions. One group of DRG neurons was treated with 25 pg (200 μL) of BoNT/A 24 h before the assay. Some wells of neuron-neutrophil co-cultures were treated with the antagonists CGRP 8-37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) at the time of neutrophil addition. For all conditions described for neutrophil opsonophagocytic killing assays, plates were incubated for 1h at 37°C with gentle shaking (150 rpm). The amount of extracellular and intracellular bacteria was determined after resuspension in ice-cold ddH 2 O by serial dilution plating on TSA plates with sheep blood agar (BD Biosciences), and bacterial colonies were counted after overnight incubation at 37°C in 5% CO 2 . The multiplication factor of net bacterial growth was calculated as the number of cfu recovered/ number of cfu added to wells.

Neutrophil myeloperoxidase activity assay Mouse neutrophils were isolated from mouse bone marrow using EasySep mouse neutrophil enrichment kit and EasySep Magnet according to manufacturer’s instructions (StemCell). These neutrophils (5x105 cells per well) were then treated with CGRP (0.01-1 μM, GenScript) or vehicle (PBS), immediately prior to mixture with S. pyogenes (5x103 cfu) M1 wt strain or vehicle (PBS) in 200 μL of neurobasal-A medium containing 10% of fresh mouse serum. Serum was obtained after coagulation of whole mouse blood. Plates were incubated for 30 min at 37°C with gently shaking (150 rpm), and supernatant collected for myeloperoxidase (MPO) activity measurements. Supernatants (50 μL) were added to 200 μL of 50 mM phosphate buffer solution (pH 6.0) containing 0.167 mg/mL of peroxidase substrate o-dianisidine dihydrochloride (Santa Cruz Biotech) and 0.05% hydrogen peroxide (Santa Cruz Biotech) and incubated for 30 min at room temperature. MPO activity was determined spectrophotometrically by measuring the increase in absorbance at 450 nm.

Lancefield assay Gryllos et al., 2008 Gryllos I.

Tran-Winkler H.J.

Cheng M.-F.

Chung H.

Bolcome 3rd, R.

Lu W.

Lehrer R.I.

Wessels M.R. Induction of group A Streptococcus virulence by a human antimicrobial peptide. 600 of 0.15 in L3 medium at 37°C with 5% CO 2 and diluted in sterile PBS. Quantitative cultures of the bacterial suspension were performed to allow precise quantification of the starting inoculum. Whole human blood (10 mL/tube) was collected into heparin-containing tubes (BD Vacutainer, Fisher Scientific) Approximately 20-200 S. pyogenes cfu were inoculated into heparinized whole blood obtained from each of three healthy donors supplemented with human CGRP (0, 10 or 100 nM) and incubated for 3 h at 37°C with end-over-end rotation. Bacterial survival was quantified as multiplication factor of number of surviving colonies relative to the starting inoculum. Each condition was tested in triplicate. Human blood phagocytosis assays (Lancefield bactericidal test) were performed as described previously with slight modifications (). S. pyogenes M1 wt strain was cultured to ODof 0.15 in L3 medium at 37°C with 5% COand diluted in sterile PBS. Quantitative cultures of the bacterial suspension were performed to allow precise quantification of the starting inoculum. Whole human blood (10 mL/tube) was collected into heparin-containing tubes (BD Vacutainer, Fisher Scientific) Approximately 20-200 S. pyogenes cfu were inoculated into heparinized whole blood obtained from each of three healthy donors supplemented with human CGRP (0, 10 or 100 nM) and incubated for 3 h at 37°C with end-over-end rotation. Bacterial survival was quantified as multiplication factor of number of surviving colonies relative to the starting inoculum. Each condition was tested in triplicate.

CGRP release assay from skin explants Skin punch biopsies (12 mm) were collected from the uninfected or infected flank skin samples of euthanized mice, and rapidly transferred to 24-well plates containing 1 mL of DMEM. Explants were incubated at 32°C with gentle shaking (150 rpm) for 30 min. After incubation, the bath supernatant from the organ cultures was collected, and assayed to determine CGRP concentration with the CGRP EIA kit (Cayman Chemical) according to manufacturer`s instructions.