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Supplemental Information Complement Protein C1q Directs Supplemental information Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells Marie E. Benoit, Elizabeth V. Clarke, Pedro Morgado, Deborah A. Fraser and Andrea J. Tenner Content of supplemental information Supplemental data: - Supplementary Figure 1 - Supplementary Figure 2 - Supplementary Figure 3 - Supplementary Table 1 1 Supplementary Figure 1. Apoptosis and C1q binding to apoptotic lymphocytes. (A-C) CD3+ lymphocytes, pre-labeled or not with a red cell membrane tracker (PKH26) (C), were incubated for 16 h in media with FBS (EAL, AnnexinV+/PI-) or without FBS (LAL, annexinV+/PI+) after γ- irradiation (10 Gy dose). Apoptosis was determined by flow cytometry using annexinV/propidium iodide staining. AL were considered in an early stage (EAL) if PI incorporation was lower than 20%. (D-F) LAL were incubated with different concentrations of C1q (0-300 g/ml) for 30 or 60 min, washed to remove unbound C1q and stained with murine monoclonal anti-human C1q. Percentage of binding (D) and mean fluorescence intensity (MFI, E) were determined by flow cytometry. Results represent mean ± SD of 2 independent experiments. (F) C1q (150 μg/ml) binding to PKH26-prelabeled EAL and LAL determined by flow cytometry.Representative FACS plots are shown. 2 Supplementary Figure 2. Phenotype of HMDMs and uptake of EAL and LAL by HMDMs.(A) Phenotype of HMDMs was assessed by flow cytometry. HMDMs showed no expression of the activation markers CD80 and CD86 or dendritic cell specific marker DC-SIGN and appropriate expression of CD40, CD54, CD14, MHCII, CD11b, CD11c and CD32, the relative expression for each marker being consistent with the literature 9;45. Representative FACS plots are shown. (B) PKH26-labeled LAL were incubated with 150 μg/ml C1q for 60 min, washed and then incubated with HMDMs at different ratio for 30, 60 or 120 min. Percentage of phagocytosis was determined by flow cytometry. Results represent mean ± SD of 2 independent experiments. (D) HMDMs were incubated with PKH26-labeled EAL and LAL, pre-incubated or not with C1q, at a 5:1 ratio for 1 h. Cells were stained with FITC-phalloidin and analyzed by confocal microscopy. Representative micrographs of 3 independent experiments are shown. Scale bar = 20 μm. 3 Supplementary Figure 3. Validation of microarray fold-changes by qRT-PCR. (A) Pearson correlation coefficient-based heat map (complete linkage method) representation of the gene intensities.(B-F)Comparison of array and qRT-PCR fold-differences for 12 significantly modulated genes by C1q in HMDMs. The results are expressed as the means of log2 fold- difference over unstimulated HMDMs. Correlation coefficient (r2) between array and qRT-PCR ratio were determined by linear regression analysis. Absolute values in fold-change obtained from microarray results and qRT-PCR results are different (since qRT-PCR is a more sensitive technique than microarray particularly for low expressed genes (see Morey JS et al., 2006; Roth CM., 2002; Canales RD et al., 2006) but every gene find up-regulated by microarray was also find up-regulated by qRT-PCR, no matter the absolute fold-change. (G) Expression of IL-27 in 4 HMDMs ± AL or AL alone (n = 1) to show absence of expression of IL-27 in AL, thus excluding any contamination by RNA from AL of our microarray studies. Supplementary Table 1. Significantly modulated genes by C1q in HMDM during the uptake of apoptotic lymphocytes See excel spreadsheet. 5 Table S1. Significantly modulated genes by C1q in HMDM during the uptake of apoptotic lymphocytes Log2 fold-difference over unstimulated HMDM Log2 fold-difference C1q over no C1q Gene assignment mRNA assignment Gene symbol Gene name LPS EAL+LPS C1q-EAL+LPS LAL+LPS C1q-LAL+LPS C1q-EAL+LPS - EAL+LPS C1q-LAL+LPS -LAL+LPS --- AB062482 // chr5 // 89 // 8 // 9 // 0 -- -- 1.566 0.720 0.342 0.755 0.670 -0.378 -0.085 --- AF090930 // chr17 // 88 // 7 // 8 // 0 -- -- 0.943 1.448 0.935 1.998 0.782 -0.512 -1.216 --- AF090933 // chr15 // 89 // 8 // 9 // 0 -- -- -1.308 -1.111 -1.628 -1.587 -1.832 -0.516 -0.245 --- AF118062 // chr5 // 100 // 23 // 23 // 0 -- -- -1.161 -1.618 -0.544 0.143 -0.216 1.074 -0.359 --- AF242527 // chr4 // 42 // 10 // 24 // 0 -- -- 1.973 0.623 0.784 1.297 1.047 0.162 -0.250 --- AF364863 // chr11 // 50 // 2 // 4 // 0 -- -- 0.606 1.367 0.884 0.889 1.380 -0.483 0.491 --- AF370400 // chr16 // 100 // 4 // 4 // 0 -- -- -0.482 -0.529 -1.794 -0.910 -0.662 -1.266 0.248 --- AF422192 // chr14 // 100 // 24 // 24 // 0 /// EN-- -- 1.575 0.845 2.030 1.364 1.691 1.185 0.327 --- AK000313 // chr7 // 100 // 24 // 24 // 0 /// EN -- -- -2.710 -0.904 -1.287 -1.659 -0.204 -0.383 1.455 --- AK022213 // chr12 // 94 // 17 // 18 // 0 -- -- 0.755 1.337 2.164 2.675 2.547 0.827 -0.128 --- AK024372 // chr16 // 100 // 9 // 9 // 0 -- -- 1.293 1.673 0.821 1.368 1.802 -0.853 0.434 --- AK024534 // chr17 // 100 // 20 // 20 // 0 -- -- -0.644 -0.636 -0.426 -2.248 -0.736 0.210 1.512 --- AK026249 // chr11 // 96 // 23 // 24 // 0 -- -- 1.693 2.333 0.742 1.110 1.731 -1.592 0.621 --- AK054879 // chr12 // 100 // 21 // 21 // 0 -- -- -0.574 -1.791 -2.175 -0.698 0.262 -0.384 0.961 --- AK055340 // chr1 // 40 // 10 // 25 // 0 -- -- -0.686 -0.559 -1.266 -0.672 0.270 -0.707 0.942 --- AK056230 // chr7 // 100 // 20 // 20 // 0 -- -- 0.553 0.517 0.691 0.693 1.244 0.174 0.551 --- AK074630 // chr8 // 100 // 7 // 7 // 0 /// ENST0-- -- 0.277 0.825 1.156 1.252 0.864 0.331 -0.388 --- AK092964 // chr3 // 100 // 25 // 25 // 0 -- -- 2.422 0.625 1.821 0.517 1.284 1.197 0.766 --- AK093576 // chr2 // 100 // 25 // 25 // 0 -- -- -0.713 -1.192 -0.186 0.009 -1.179 1.006 -1.188 --- AK094874 // chr12 // 100 // 25 // 25 // 0 /// EN-- -- -0.735 -1.298 -0.678 -2.413 -0.533 0.620 1.880 --- AK096781 // chr6 // 100 // 20 // 20 // 0 -- -- 1.145 1.158 0.689 1.852 1.843 -0.469 -0.010 --- AK098314 // chr15 // 75 // 18 // 24 // 0 /// AK -- -- 0.177 0.733 0.990 1.506 1.190 0.258 -0.316 --- AK098573 // chr18 // 100 // 12 // 12 // 0 -- -- -0.639 -0.224 -0.557 -0.214 -1.465 -0.333 -1.251 --- AK128486 // chr5 // 92 // 24 // 26 // 0 -- -- 0.188 1.177 1.364 1.024 1.222 0.187 0.198 --- AY336750 // chr10 // 100 // 25 // 25 // 0 -- -- 1.937 1.665 1.307 1.845 2.029 -0.358 0.183 --- AY336750 // chr10 // 100 // 25 // 25 // 0 -- -- 1.642 1.107 1.865 2.525 1.043 0.758 -1.482 --- AY358211 // chr19 // 100 // 7 // 2 // 0 -- -- 0.246 2.938 2.298 2.523 1.710 -0.639 -0.813 --- AY358728 // chr2 // 100 // 15 // 15 // 0 -- -- 1.777 2.110 2.002 1.256 1.062 -0.108 -0.194 --- AY371029 // chr3 // 42 // 10 // 24 // 0 /// AY37-- -- 0.911 1.191 1.033 1.797 0.852 -0.159 -0.945 --- AY510107 // chr2 // 72 // 21 // 29 // 0 /// ENST-- -- 0.530 2.513 2.353 2.436 2.385 -0.160 -0.050 --- BC008667 // chr20 // 100 // 10 // 10 // 0 -- -- 2.979 2.643 1.893 2.241 2.100 -0.751 -0.141 --- BX537598 // chr5 // 100 // 25 // 25 // 0 -- -- 1.637 0.749 1.836 1.367 2.944 1.088 1.577 --- CR611676 // chr1 // 100 // 22 // 22 // 0 /// CR6-- -- 0.616 1.397 1.010 1.302 1.612 -0.387 0.310 --- ENST00000237841 // chr12 // 100 // 12 // 12 /-- -- 0.795 0.319 0.881 0.376 1.441 0.562 1.064 --- ENST00000245183 // chr9 // 71 // 5 // 7 // 0 //-- -- 1.020 1.130 1.018 1.215 1.123 -0.112 -0.092 --- ENST00000262637 // chr19 // 100 // 13 // 13 /-- -- -0.840 -0.462 -0.658 -1.816 -0.731 -0.197 1.084 --- ENST00000272567 // chr2 // 100 // 12 // 12 // -- -- -0.440 0.229 0.207 0.928 1.828 -0.021 0.899 --- ENST00000302153 // chr17 // 100 // 11 // 11 /-- -- 0.523 0.627 1.071 0.553 1.270 0.445 0.717 --- ENST00000303310 // chr6 // 100 // 17 // 17 // -- -- 0.549 1.035 0.323 1.669 0.746 -0.712 -0.923 --- ENST00000306515 // chr2 // 100 // 17 // 17 // -- -- 0.642 0.934 1.014 1.233 2.965 0.081 1.731 --- ENST00000309874 // chr15 // 100 // 17 // 17 /-- -- 0.874 0.954 0.660 1.464 3.145 -0.294 1.682 --- ENST00000312918 // chr2 // 100 // 21 // 21 // -- -- 1.381 0.840 0.974 0.787 -0.040 0.134 -0.827 --- ENST00000313192 // chr7 // 100 // 17 // 17 // -- -- -0.171 0.699 1.150 1.782 1.448 0.451 -0.334 --- ENST00000313760 // chr2 // 100 // 17 // 17 // -- -- 1.405 0.676 0.122 0.218 0.091 -0.554 -0.127 --- ENST00000316517 // chr11 // 100 // 22 // 22 /-- -- 1.519 1.078 1.845 0.406 1.631 0.767 1.225 --- ENST00000325243 // chr11 // 100 // 19 // 19 /-- -- 0.743 2.229 1.307 1.432 1.069 -0.922 -0.362 --- ENST00000326513 // chr12 // 100 // 25 // 25 /-- -- 0.141 1.019 0.666 1.721 0.265 -0.353 -1.456 --- ENST00000326910 // chr11 // 100 // 25 // 25 /-- -- 0.095 0.474 0.027 1.774 0.642 -0.446 -1.132 --- ENST00000327881 // chr19 // 100 // 22 // 22 /-- -- 1.710 0.345 0.758 0.618 1.821 0.413 1.202 --- ENST00000328207 // chr12 // 100 // 8 // 8 // 0-- -- -0.562 -0.855 0.463 -2.832 -0.069 1.317 2.763 --- ENST00000330381 // chr11 // 100 // 25 // 25 /-- -- -0.296 0.951 1.599 1.940 1.591 0.648 -0.349 --- ENST00000331736 // chr3 // 100 // 14 // 14 // -- -- 0.312 0.270 0.623 1.598 0.862 0.353 -0.735 --- ENST00000333085 // chr5 // 100 // 16 // 16 // -- -- -0.796 -1.382 -0.996 -1.752 -2.281 0.387 -0.529 --- ENST00000343867 // chr8 // 100 // 23 // 23 // -- -- -0.689 0.708 0.279 1.645 -0.098 -0.430 -1.743 --- ENST00000355745 // chr15 // 100 // 23 // 23 /-- -- 0.554 0.777 0.075 1.653 0.899 -0.702 -0.754 --- ENST00000357418 // chr16 // 100 // 10 // 10 /-- -- 1.021 1.586 0.921 0.892 0.589 -0.665 -0.303 --- ENST00000357646 // chr16 // 100 // 9 // 9 // 0-- -- 1.056 1.611 0.952 0.947 0.695 -0.659 -0.253 --- ENST00000358601 // chr19 // 100 // 24 // 24 /-- -- 0.558 0.569 0.114 0.855 1.757 -0.455 0.902 --- ENST00000362367 // chr12 // 100 // 25 // 25 /-- -- 0.163 0.243 0.190 0.441 1.405 -0.054
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