Supplementry figure 1 Igf 1 mrna 5 15 1 5 Arg1 mrna 16 1 8 Col11 mrna 5 3 1 Mmp 13 mrna 15 1 5 Tslp mrna 3 1 Il5 mrna 3 1-1 Il33 mrna 6 Figure 1s. Kinetis of woun heling ftors n erly Th-type response ytokines fter inoultion with N. rsiliensis. Gene expression of insulin- like growth ftor (Igf1), Arg1, Collgen, type I, lph 1 (Col11), mtrix metlloproteinse 13 (Mmp13), Tslp, Il5, n Il33 ws etermine y rel time PCR. Gene expression is expresse s fol inrese over nïve ontrols fter normliztion to 18sRNA. Dt shown re the men n SEM from five iniviul mie per group n re representtive of two inepenent experiments.
Supplementry figure 1 N 1 N 11.7. 3 N 5 N N 5 N 3 N 1 6 8 Ly6G 3 N 6.3 5 N 5.1 N 5 N 3 N 1 1 3 8 1 BAL RBC ( 1 7 ) BAL neu ( 1 ) N 5 N 3 N 1 8 1 Il17 mrna (tr/untr) N 5 N 3 N 1 1 3 Il13 mrna (tr/untr) 1 Arg1 mrna (tr/untr) Figure s. Effets of N. rsiliensis ose on lung mge n ytokine gene expression. Mie were inoulte with 1, 3, n 5 N. rsiliensis (N) L3. At y fter inoultion, lungs were ollete, exmine histologilly (Sle r, 5µm) n sore for ALI () n neutrophils (Ly6G + ) were ssesse y FACS nlysis (). BAL RBCs n BAL neutrophils were ounte n lung Il17 mrna expression etermine y rel time RT-PCR (). At y fter inoultion, lung Il13 n Arg1 mrna were ssesse y rel time RT-PCR (). All t shown re the men n SEM from five iniviul mie per group n re representtive of two inepenent experiments. p<.1.
Supplementry figure 3 Anti Ly6G Anti Ly6G Ilr / Ilr / Il17r / Anti IL-17A Il17r / Anti IL-17A 6 81 6 81 6 81 e Ilr / f Ilr / + N + N Ilr / Anti IL-17A..6 1. Enhne puse (penh) Anti IL-17A Figure 3s. Assessment of lung mge n impire lung funtion in N. rsiliensis-inoulte mie. ( e) Imges of representtive H&E stine tissue setions n ute lung injury (ALI) sore of lung tissue from N. rsiliensis (N)-inoulte KO mie or inoulte mie trete with epleting or loking As t y ( ) or y (, e) fter N. rsiliensis (N) inoultion (Sle r, 5µm). (f) irwy hyperresponsiveness (seline mesurement) inite s penh (puse enhne) vlues t y. In ll experiments, histologil (H&E) preprtions re representtive of setions from five nimls/tretment groups while mens n SEM re of five mie/group. Experiments shown were repete two times with similr results. p<.5, p<.1. 8 1 6 81
Supplementry figure Anti Ly6G (1A8) Anti Ly6G (1A8) Il17r +/+ Il17r / Ly6G(1A8) 8.6 71..97 99.3 Gr-1(8C5).3 99.97 Ly6G(1A8) 8.6 71.7 8. 91.56 Anti IL-17A Ly6G(1A8) 9.3 69.17 18.36 81.6 Anti Ly6G + N + N Anti Ly6G 6 8 Bleeing time (seon) Figure s. Neutrophils in lung re eplete in N. rsiliensis-inoulte mie ministere Ly6Gspeifi ntioy, reue in Il17r / mie or mie ministere IL-17A-speifi A n neutrophil epletion oes not ffet loo ogultion. ( ): Lung ells from mie ministere Ly6Gspeifi ntioy n isotype ontrol (), or lung ells from Il17r / n ontrols (), n mie ministere IL-17A-speifi ntioy (), were isolte t y fter inoultion with N. rsiliensis (N), stine with Ly6G-speifi, or Gr-1-speifi ntioy, n nlyze y flow ytometry. FACS nlyses re of poole lung ells from five iniviul mie per group n re representtive of two inepenent experiments. () mie were trete s in, n loo ogultion ws ssesse. All t re expresse s the men n SEM of five nimls/tretment group n similr results were otine in two inepenent experiments.
Supplementry figure 5 (D) Anti IGF-1R (D) (D5) Il1 / (D5) Anti IGF-1R 3 6 9 Il1 / 6 8 1.% BEC + N N 1 3 Arginse tivity (U mg 1 ) PBS (D6).%BEC (D6).% BEC PBS 6 8 Figure 5s. Lung mge s ssesse y histology n BEC inhiition of rginse tivity. ( ) H&E stining of lung tissue setions n ute lung injury (ALI) soring in: () N. rsiliensis-inoulte mie (y ) trete with IGF-1R-speifi ntioy or isotype ontrol; () N. rsiliensis-inoulte Il1 / or mie (y 5); () N. rsiliensis inoulte mie (y 6) ministere BEC or PBS vehile. Stine lung tissue setions re representtive exmples of results from five mie/group (Sle r, 5 µm) n s re expresse s the men n SEM of five mie/group. () Arginse iotivity in lung tissue ws etermine in N. rsiliensis (N)-inoulte mie (y 6) ministere BEC s esrie in the Methos. Dt re expresse s the men n SEM of four nimls/tretment group. All experiments were repete twie with similr results. p<.5, p<.1.
Supplementry figure 6 BAL - m ( 1 5 ). 1.5 1..5. BAL 88.3 Lung 17.8 Lung 1.9 Arg1 Mmp13 Igf1 Il1 65. 15.5 13.6 N, 7 N, 3 mrna 9 18 7 6 8...8 1. (tr/untr) F/8 7. 1.7 Sigle F 8. N DT+N M + DT + N DT + N N.5 16. 16.3 M+ DT+N 1 3 5 Lung F/8 + ells ( 1 6 ) CCR3
Figure 6S: Mrophges show inrese expression of woun heling ftors n flow ytometri nlyses of lung tissue n BAL mrophges from DT-trete CD11-DTR mie. () BAL ws ollete t ys,,, n 7 fter inoultion with N. rsiliensis (N) n mrophge (F/8 + ) numer etermine y flow ytometri nlyses. () F/8 + mrophges were sorte t y (nïve mie), 3, n 7 fter N. rsiliensis (N)-inoultion n ssesse for Il1, Arg.1, Mmp13, n Igf1 mrna using rel time RT-PCR. () CD11DTR mie were inoulte with N. rsiliensis (N) n ministere DT to eplete mrophges in vivo. lung-erive mrophges were trnsferre to DT trete CD11DTR mie prior to inoultion. On y 3 fter inoultion, BAL n lung tissue single ell suspensions were stine for mrophges (F/8 + ) n eosinophils (siglef + n CCR3 + ) n nlyze y flow ytometry. Dt re from poole smples from five mie/tretment group n similr results were otine in two inepenent experiments. () Totl lung ells were ounte n lung F/8 + mrophges were lulte from perentge of totl. Results re reporte from three to five iniviul mie per group n similr results were otine in two inepenent experiments. p<.5.
Supplementry figure 7. Cloro-lipo + N PBS-lipo + N 3. 5 115 5 1 15 BAL RBC ( 1 6 ) BAL neu ( 1 ) 6 8 1 3 6 8 1 Arg1 mrna Mmp13 mrna Igf1 mrna 8 6 PBS lipo + N Cloro lipo + N N: + + PBS lipo: + Cloro lipo: +. PBS lipo + N Cloro lipo + N 7.3 67.5 5. BAL F/8 1. 15. 11.5 Lung
Figure 7S: Lung mge s ssesse y Cloronte-liposome epletion of mrophges. () Mie were trete with Cloronte-liposomes (Cloro-lipo) or PBS-liposomes (PBS-lipo) n inoulte with N. rsiliensis (N). At y, BAL RBCs n BAL neutrophils were ounte n lung Arg1, Igf1, n Mmp13 mrna expression etermine y rel time RT-PCR. () Stine lung tissue setions re representtive exmples of results from five mie/group (Sle r, 5 µm) n ALI sores re expresse s the men n SEM of five mie/group. () Flow ytometri nlyses of F/8 + mrophges in N. rsiliensis (N)-inoulte mie ministere PBS-liposomes or loronteliposomes. Similr results were otine in three inepenent experiments. p<.5, p<.1.