Table S1: Primers used in this study. Gene Species Forward primer Reverse primer Reference Gapdh Mouse AACGACCCCTTCATTGAC TCCACGACATACTCAGCAC (1) Ciita Mouse ACGCTTTCTGGCTGGATTAGT TCAACGCCAGTCTGACGAAGG (1) H2-DMb Mouse CAACAAGGAGAAGACGGCTCA CGCTGTGCTGAACCACG (1) Arg1 Mouse CAGAAGAATGGAAGAGTCAG CAGATATGCAGGGAGTCACC (2) Nos2 Mouse GGCAGCCTGTGAGACCTTTG GCATTGGAAGTGAAGCGTTTC RTPrimerDB ID: 3297 (3) Il10 Mouse AGAGAAGCATGGCCCAGAAATC TCATGGCCTTGTAGACACCTTG Designed with Primer3 Il12b Mouse AGAAAGGTGCGTTCCTCGTAG AGCCAACCAAGCAGAAGACAG Designed with Primer3 Il2 Mouse CCTGAGCAGGATGGAGAATTACA TCCAGAACATGCCGCAGAG RTPrimerDB ID: 135 (3) Ifng Mouse TCAAGTGGCATAGATGTGGAAGAA TGGCTCTGCAGGATTTTCATG RTPrimerDB ID: 146 (3) Tbx21 Mouse CCTCTTCTATCCAACCAGTATC CTCCGCTTCATAACTGTGT (4) Il4 Mouse AGATGGATGTGCCAAACGTCCTCA AATATGCGAAGCACCTTGGAAGCC (5) Il17a Mouse ATCCCTCAAAGCTCAGCGTGTC GGGTCTTCATTGCGGTGGAGAG (6) Il22 Mouse ATCGTCAACCGCACCTTTAT GACTCCTCGGAACAGTTTCTC (7) Mrc1 Mouse GCAAATGGAGCCGTCTGTGC CTCGTGGATCTCCGTGACAC (8) Retnla Mouse CCCAGGATGCCAACTTTGAA GGCCCATCTGTTCATAGTCT (8) Chi3l3 Mouse GGGCATACCTTTATCCTGAG CCACTGAAGTCATCCATGTC (8) GAPDH Human GACCTGACCTGCCGTCTA GTTGCTGTAGCCAAATTCGTT (9) IL10 Human GACTTTAAGGGTTACCTGGGTTG TCACATGCGCCTTGATGTCTG PrimerBank ID: 24430216c1 (10) IL12B Human GGAGTACCCTGACACCTGGA ACGCTAATGCTGGCATTTTT (11)

Table S1 References 1. Pai RK, Pennini ME, Tobian AA, Canaday DH, Boom WH, Harding CV. 2004. Prolonged toll-like receptor signaling by Mycobacterium

tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages. Infect. Immun. 72:6603-6614.

2. Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Clement M, Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A, Caligiuri G. 2010. Macrophage plasticity in experimental atherosclerosis. PLoS ONE 5:e8852.

3. Pattyn F, Speleman F, De Paepe A, Vandesompele J. 2003. RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res 31:122-123.

4. Ariga H, Shimohakamada Y, Nakada M, Tokunaga T, Kikuchi T, Kariyone A, Tamura T, Takatsu K. 2007. Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals. Immunology 122:210-221.

5. Stellato C, Gubin MM, Magee JD, Fang X, Fan J, Tartar DM, Chen J, Dahm GM, Calaluce R, Mori F, Jackson GA, Casolaro V, Franklin CL, Atasoy U. 2011. Coordinate regulation of GATA-3 and Th2 cytokine gene expression by the RNA-binding protein HuR. J Immunol 187:441-449.

6. Smith E, Stark MA, Zarbock A, Burcin TL, Bruce AC, Vaswani D, Foley P, Ley K. 2008. IL-17A inhibits the expansion of IL-17A-producing T cells in mice through "short-loop" inhibition via IL-17 receptor. J Immunol 181:1357-1364.

7. Chellan B, Yan L, Sontag TJ, Reardon CA, Hofmann Bowman MA. 2014. IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1. J Lipid Res 55:443-454.

8. Van den Bossche J, Lamers WH, Koehler ES, Geuns JM, Alhonen L, Uimari A, Pirnes-Karhu S, Van Overmeire E, Morias Y, Brys L, Vereecke L, De Baetselier P, Van Ginderachter JA. 2012. Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes. J Leukoc Biol 91:685-699.

9. Hardy GA, Sieg SF, Rodriguez B, Jiang W, Asaad R, Lederman MM, Harding CV. 2009. Desensitization to type I interferon in HIV-1 infection correlates with markers of immune activation and disease progression. Blood 113:5497-5505.

10. Spandidos A, Wang X, Wang H, Seed B. 2010. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res 38:D792-799.

11. Mogilenko DA, Kudriavtsev IV, Trulioff AS, Shavva VS, Dizhe EB, Missyul BV, Zhakhov AV, Ischenko AM, Perevozchikov AP, Orlov SV. 2012. Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages. J Biol Chem 287:5954-5968.

Time (min): 0 1 5 10 15 30 45 60 90 120 150 180 24075

Wild-type, M. tuberculosis-infected

ERK

,ț%Į

p-ERK

Figure S1. M. tuberculosis infection of macrophages induces sustained ERK signaling and transient

1)�ț%�VLJQDOLQJ�

Macrophages were infected with M. tuberculosis for the indicated times in minutes, and cell lysates

were prepared and analyzed by Western blotting. ERK phosphorylation (“p-ERK”) peaked at 30 min

RI�LQIHFWLRQ�DQG�ZDV�VXVWDLQHG�DW�D�OHYHO�DERYH�EDVHOLQH�RXW�WR�DW�OHDVW���K���,Q�FRQWUDVW��1)�ț%�VLJQDOLQJ��,ț%Į�GHJUDGDWLRQ��ZDV�PD[LPL]HG�DW����PLQ�DQG�UHFRYHUHG�WR�EDVHOLQH���7KH�H[DFW�NLQHWLFV�of activation of these signaling pathways may depend on the time of contact of M. tuberculosis

bacteria with macrophages; for a more synchronized stimulation using a soluble TLR2 ligand,

UHFRPELQDQW�/SU*��VHH�)LJXUH��%�

Figure S2. The ERK pathway has a unique role in regulating the balance of IL-10 and IL-12 secretion

by M. tuberculosis-infected macrophages.

0DFURSKDJHV�ZHUH�SUH�WUHDWHG�ZLWK�LQKLELWRUV��LQGLFDWHG�E\���V\PEROV��DW����ȝ0�ILQDO�FRQFHQWUDWLRQ�RU�DMSO vehicle control, and then infected with M. tuberculosis (“Mtb”) or left uninfected for 24 h.

Supernatants were collected and analyzed for cytokine secretion by ELISA following the

PDQXIDFWXUHUV�LQVWUXFWLRQV��5'�6\VWHPV����7KH�GDWD�UHSUHVHQW�WZR�LQGHSHQGHQW�H[SHULPHQWV�DQG�show the means ± SEM. Statistical significance was determined using one-way ANOVA

(****, P<0.0001, **: P<0.01, NS: P>0.05).

7KHVH�H[SHULPHQWV�GLG�QRW�UHYHDO�D�UROH�IRU�-1.��XVLQJ�WKH�LQKLELWRU�63��������LQ�FRQWUROOLQJ�PDFURSKDJH�F\WRNLQH�H[SUHVVLRQ�IROORZLQJ�M. tuberculosis infection. Inhibition of ERK (by U0126) or

S���0$3.��E\�6%��������UHYHDOHG�VLJQLILFDQW�GRZQUHJXODWLRQ�RI�,/����VHFUHWLRQ��)LJ��6�$���EXW�RQO\�(5.�LQKLELWLRQ�UHVXOWHG�LQ�VLJQLILFDQW�XSUHJXODWLRQ�RI�,/����S����)LJ��6�%���LQGLFDWLQJ�WKDW�WKH�UHYHUVDO�of the macrophage cytokine profile to favor production of pro-inflammatory IL-12 is controlled uniquely

by ERK.

0

100

200

300IL

-10

(pg/

ml)

SB203580SP600125

U0126Mtb

- - + + - - - -- - - - + + - -- - - - - - + +- + - + - + - +

* * * *NS

0

100

200

300

400

IL-1

2 p7

0 (p

g/m

l)

SB203580SP600125

U0126Mtb

- - + + - - - -- - - - + + - -- - - - - - + +- + - + - + - +

NS NS ****

A %

0

100

200

300

IL-1

2 p7

0 (p

g/m

l)

Tpl2LPSP3C

+/+ -/- +/+ -/-+ + - -- - + +

NS

****

0

100

200

300

400IL

-10

(pg/

ml)

Tpl2LPSP3C

+/+ -/- +/+ -/-+ + - -- - + +

**** ****

Figure S3. TLR4 activation by lipopolysaccharide (LPS) does not regulate macrophage IL-10 and

IL-12 cytokine balance in the same fashion as TLR2 activation.

Macrophages were treated with 10 ng/ml purified E. coli LPS, a TLR4 stimulus, or 10 nM synthetic

Pam3CSK

4 (“P3C”), a TLR2 stimulus, for 24 h. Supernatants were then collected and analyzed by

ELISA according to the manufacturer's instructions (R&D Systems). Data represent two independent

H[SHULPHQWV�DQG�VKRZ�PHDQV���6(0���6WDWLVWLFDO�VLJQLILFDQFH�ZDV�GHWHUPLQHG�XVLQJ�6WXGHQWV�t test

(****: P<0.0001, NS: P>0.05).

LPS induced less IL-10 from macrophages than Pam3CSK

4 (Fig. S3A). IL-10 induction by LPS was

partially dependent on Tpl2, but less Tpl2-dependent than IL-10 induced by Pam3CSK

4 (Fig. S3A).

,/����S���H[SUHVVLRQ�ZDV�QRW�LQGXFHG�E\�/36�LQ�ZLOG�W\SH�RU�Tpl2-/- macrophages, while Pam3CSK

4

LQGXFHG�KLJK�OHYHOV�RI�,/����S���H[SUHVVLRQ�LQ�Tpl2-/-�PDFURSKDJHV��)LJ��6�%����7KHVH�UHVXOWV�LQGLFDWH�that the TLR2 signaling pathway is unique in controlling the levels of IL-10 and IL-12, and regulates

these cytokines through Tpl2 and ERK.

A %

Figure S4. Other genes typically associated with the M2 macrophage polarization state, which are

induced by IL-4 signaling, are not components of the macrophage response to M. tuberculosis.

Macrophages were treated with IL-4 or infected with M. tuberculosis (“Mtb”) for 24 h, and gene

H[SUHVVLRQ�ZDV�GHWHUPLQHG�E\�T57�3&5��M. tuberculosis�LQIHFWHG�PDFURSKDJHV�GLG�QRW�H[SUHVV�Mrc1 (Fig. S4A), Retnla��)LJ��6�%���RU�Chi3l3 (Fig. S4C), in contrast to IL-4-treated M2 macrophages.

'DWD�DUH�PHDQV���6(0�DQG�UHSUHVHQW�WZR�LQGHSHQGHQW�H[SHULPHQWV�� ��3�������� ��3�������1'��not detectable.

0.0

0.1

0.2

0.3

0.4

IL-4Mtb

- + -- - +

* *

* * *

Mrc1/Gapdh

0.0

0.1

0.2

0.3

0.4

IL-4Mtb

- + -- - +

ND ND

Retnla/Gapdh

0.0000

0.0005

0.00100.3

0.4

0.5

IL-4Mtb

- + -- - +

* * *

* * *

Chi3l3/Gapdh

A B C