Lymphotoxin β Activation on Macrophages Induces Cross-Tolerance to TLR4 and TLR9 Ligands

This information is current as Nadin Wimmer, Barbara Huber, Nicola Barabas, Johann of September 27, 2021. Röhrl, Klaus Pfeffer and Thomas Hehlgans J Immunol 2012; 188:3426-3433; Prepublished online 22 February 2012; doi: 10.4049/jimmunol.1103324

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lymphotoxin b Receptor Activation on Macrophages Induces Cross-Tolerance to TLR4 and TLR9 Ligands

Nadin Wimmer,* Barbara Huber,* Nicola Barabas,* Johann Ro¨hrl,* Klaus Pfeffer,† and Thomas Hehlgans*

Our previous studies indicated that lymphotoxin b receptor (LTbR) activation controls and downregulates inflammatory reac- tions. In this study, we report that LTbR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30a, which negatively regulates NF-kB activation induced by TLR signaling. LTbR activation results in a downregulation of proinflammatory and mediator expression upon TLR restimulation, demonstrating that LTbR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30a-specific small interfering RNA abolished the LTbR-dependent induction of TRIM30a and LTbR-mediated TLR cross-tolerance. Concordantly,

LTbR activation on -derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Further- Downloaded from more, we have generated cell type-specific LTbR-deficient mice with ablation of LTbR expression on macrophages/neutrophils (LTbRflox/flox 3 LysM-Cre). In bone marrow-derived macrophages derived from these mice LTbR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTbR expression on macrophages (LTbRflox/flox 3 LysM-Cre) are resistant to LTbR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTbR activation on macrophages by T cell-derived lymphotoxin a1b2 controls proinflammatory responses by activation of a TRIM30a- controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions. The Journal of Immu- http://www.jimmunol.org/ nology, 2012, 188: 3426–3433.

nflammation is a complex pathophysiological condition ini- and cells of the myeloid lineage (7, 8). So far, most studies have tially mediated primarily by innate immune cells in response focused on the critical role of LTbR signaling in the development I to and/or tissue damage (1, 2). Innate immune cells and maintenance of secondary lymphoid organ integrity (9–11) detect and respond to danger signals such as pathogens and/or and the control of -mediated immune homeostasis tissue damage by activating their TLRs on their cell surface. (12, 13). Furthermore, some reports have demonstrated a critical However, chronic and repeated stimulation through TLRs renders role for LTbR signaling for the protection against Citrobacter by guest on September 27, 2021 immune cells hyporesponsive to subsequent stimulation, a phe- rodentium-induced colitis (14, 15) and Mycobacterium tubercu- nomenon known as TLR tolerance (3). The activation of innate losis, Listeria monocytogenes (16), as well as cytomegalovirus immune cells triggers a robust but essential inflammatory re- (17) . Recent results have shown that ablation of LTbR sponse that needs to be tightly regulated (4, 5). Uncontrolled in- signaling using either a functional inhibitor of LTbR activation flammatory reactions lead to extensive tissue damage and the (LTbR:Ig) or LTbR-deficient mice or mice deficient for the T cell- manifestation of pathophysioplogical conditions such as chronic derived ligand LTb results in a significant aggravation of inflam- inflammation, , and autoimmune disease (6). Membrane- mation (18). Activation of the LTbR by its membrane-associated anchored lymphotoxin (LT)a1b2 and LIGHT, both members ligand LTab, but not LIGHT, seems to be crucial for the down- of the TNF superfamily, are functional ligands for the LTb re- regulation of the inflammatory response (19). However, the cel- ceptor (LTbR). Both ligands are expressed only on activated lym- lular and molecular mechanisms underlying this protective role of phocytes, NK cells, and a subset of follicular B cells, whereas LTbR activation have so far not been elucidated. the LTbR is primarily expressed on epithelial and stromal cells In this study we have identified tripartite motif containing (TRIM) 30a, a negative regulator of TLR-induced NF-kB activa- tion, as a target of LTbR signaling in bone marrow-derived *Institute of Immunology, University of Regensburg, 93053 Regensburg, Germany; and †Institute of Medical Microbiology and Hospital Hygiene, University of Du¨ssel- macrophages (BMDM). LTbR-induced TRIM30a expression in- dorf, 40225 Du¨sseldorf, Germany hibits the production of proinflammatory and media- Received for publication November 18, 2011. Accepted for publication January 20, tors upon TLR4 and TLR9 restimulation, demonstrating, to our 2012. knowledge, for the first time that LTbR activation induces cross- This work was supported by Deutsche Forschungsgemeinschaft Grants HE3116/5 tolerance in TLR-induced cytokine and proinflammatory medi- and HE3116/8 (to T.H.). ator production. Address correspondence and reprint requests to Prof. Thomas Hehlgans, Institute of Furthermore, we have generated cell type-specific LTbR-defi- Immunology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Re- gensburg, Germany. E-mail address: [email protected] cient mice with ablation of LTbR expression on macrophages/ flox/flox 3 The online version of this article contains supplemental material. neutrophils (LTbR LysM-Cre). BMDM derived from these Abbreviations used in this article: BMDM, bone marrow-derived macrophage; mice are resistant to TLR4 and TLR9 tolerance in vitro. Addi- BMDN, bone marrow-derived neutrophil; ES, embryonic stem; LT, lymphotoxin; tionally, no tolerance could be induced in the model of TLR4- LTbR, lymphotoxin b receptor; RPEC, resident peritoneal exudate cell; siRNA, induced cytokine production in vivo. Collectively, our data suggest small interfering RNA; TRIM, tripartite motif containing. that cell-type specific LTbR signaling is critically involved in the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 regulation of innate inflammatory immune reactions. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103324 The Journal of Immunology 3427

Materials and Methods and rechallenged with 100 ng/ml LPS or 1 mM CpG (Metabion, Mar- Generation of LTbR floxed mice tinsried, Germany) for 8 h or 200 ng/ml LPS and 20 ng/ml mouse IFN-g (AbD Serotec, Kidlington, U.K.) for 24 h. TNF and IL-6 were measured in A genomic clone derived from a mouse 129Sv/J genomic library encom- the supernatants by ELISA. Nitrite accumulation in the cell culture passing the complete coding sequence for the LTbR has been de- supernatants was measured by using the Griess assay. scribed earlier (9). This clone was modified by introducing an FRT-flanked On days 25 and 23 mice were i.p. pretreated with PBS, LPS neomycin resistance cassette followed by a loxP signal at the 39 end into (, 0127:B8; Sigma-Aldrich) (50 mg/kg or 20 mg/kg), rat the EcoRV restriction site within the 59 untranslated region of the LTbR IgG (100 mg/mouse), or agonistic anti-mLTbR mAb (100 mg/mouse). On gene. Additionally, a second loxP site had been introduced into the second day 0 the mice were challenged with LPS at a dose of 10 mg/mouse. Serum intron of the LTbR gene. was collected 1 h later and TNF was measured by ELISA. For analysis of E14.1 embryonic stem (ES) cells were transfected with linearized LTbR the TRIM30a expression in vivo, mice were killed on day 22, resident targeting vector as described (20). G418-resistant ES cell colonies were peritoneal exudate cells (RPEC) were obtained by lavage of the perito- picked. Homologous recombination was screened by PCR and subse- neum with 10 ml ice-cold RPMI 1640 containing 10% FCS. quently confirmed by genomic Southern blotting after digest of the ES cell DNA with EcoR1 and hybridization of the flanking probe, located 59 of the RNA isolation and quantitative RT-PCR targeting vector in the genomic locus. Location and orientation of both Total RNA from cultured cells was extracted by using the NucleoSpin RNA loxP sites and the FRT flanked neomycin resistance cassette were verified II (Macherey-Nagel, Du¨ren, Germany) according to the manufacturer’s by cloning of the corresponding PCR products and subsequent sequence instructions. RNA was transcribed using the Promega (Mannheim, Ger- analysis. Single integration of the targeting vector was verified by Southern many) reverse transcription system following the manufacturer’s recom- probing with the neomycin resistance cassette. Chimeric mice were gen- mendations. Quantification of mouse TRIM30a mRNA was performed erated as described (20). Deletion of the neomycin resistance cassette using an iQ multicolor real-time PCR detection system (Bio-Rad, Mu¨n- in vivo was achieved by breeding chimeric mice with FLPe deleter chen, Germany) following the manufacturer’s recommendations. For Downloaded from mice (21). Deletion of the neomycin resistance cassette was verified by standardization, 18S RNA was amplified. Primers specific for TRIM30a Southern blotting. Mice were housed in an animal facility with barrier were purchased from SABiosciences (Frederick, MD) following the conditions. For genotyping by PCR the following primers were used: 59- manufacturer’s recommendations. GAAGCATAGCATTGTCCCACGG-39 and 59-CTATGAGGCAATGGG- GAAAGAGGG-39. Western blotting Mice Analysis of TRIM30a expression was done using a polyclonal rabbit anti-TRIM30a antiserum generated by immunization of keyhole http://www.jimmunol.org/ Female C57BL/6 mice (wild-type) were obtained from Janvier (Le Genest, limpet hemocyanin-conjugated TRIM30a peptide (LHSQIKQNVLFQ) France). LTbR-deficient mice (LTbR2/2), LTab-deficent mice (LTab2/2), 2/2 synthesized by BioGenes (Berlin, Germany), as well as peroxidase- and LIGHT-deficient mice (LIGHT ) have been described previously conjugated goat anti-rabbit IgG (whole molecule) Ab (Sigma-Aldrich). (9, 22–25). MyD88-deficient mice (MyD882/2) and TNF-deficient mice 2/2 The detection of b-actin was performed with a peroxidase-conju- (TNF ) were housed at the animal facility of the University of gated chicken anti-mouse b-actin (Santa Cruz Biotechnology, Santa Regensburg and have been described previously (26, 27). Mice used for Cruz, CA). experiments were age, sex, and weight matched. ELISA In vitro stimulation assays Mouse TNF, IL-6, and IFN-g levels were measured with ELISA kits (R&D The mouse macrophage cell line J774 (ATCC TIB-67) was maintained in DuoSets; R&D Systems, Wiesbaden, Germany) according to the manu- by guest on September 27, 2021 humidified 5% CO2 at 37˚C in RPMI 1640 supplemented with 10% heat- facturer’s protocol. inactivated FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. BMDM were generated as described previously (28). Stimulations were performed NO production at day 7 in triplicates (1 3 106 cells/ml) using 10 mg/ml agonistic rat anti- mLTbR mAb (clone 5G11b, IgG2a; Hycult Biotech, Uden, The Nether- The NO production was determined using a Griess assay as described lands), 10 mg/ml rat IgG (Sigma-Aldrich, Taufkirchen, Germany), or 100 previously (30). The samples were analyzed in triplicates. ng/ml LPS from Salmonella enterica serotype abortus equi (Sigma- Aldrich, Hamburg, Germany) for the indicated times. Naive CD4+ Statistics + T cells were purified from single-cell suspensions of using a CD4 Statistical analysis was performed using the Student t test. Data are T cell isolation kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany). 6 , . + expressed as mean SD, and p 0.05 was considered statistically sig- Reanalysis demonstrated a purity of 94% (data not shown). CD4 T cells nificant. were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma- Aldrich) for 14 h, additionally treated for 4 h with 10 mg/ml mLTbR-Ig or left unstimulated. T cells were washed three times with RPMI 1640 and Results cocultured in increasing ratios (1:1, 1:5, and 1:10) with BMDM for 24 h in Generation of cell type-specific LTbR-deficient mice RPMI 1640 (Sigma-Aldrich) supplemented with 10% inactivated FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. To study the cellular and molecular mechanisms underlying the protective role of LTbR activation against an exacerbated Flow cytometry inflammatory reaction, we generated macrophage/neutrophil- Expression of LTbR was detected by FACS analysis (BD LSR II; BD specific LTbR-deficient mice by crossing LTbRfloxflox mice with Biosciences, San Jose, CA) using a specific rat anti-mLTbR biotin- LysM-Cre mice (Fig. 1). To inactivate the LTbR cell type spe- conjugated Ab (clone 3C8, IgG1; eBioscience, San Diego, CA) or an ir- relevant isotype-matched rat IgG as negative control followed by an cifically we made use of both the Cre/loxP and FRT/FLPe tech- allophycocyanin-conjugated streptavidin Ab. nology (21, 31). In the LTbR targeting vector encompassing the entire genomic LTbR gene locus, a 59 loxP site was inserted into Small interfering RNA experiments the second intron of the LTbR gene (Supplemental Fig. 1). Ad- J774 cells were transiently transfected with 1 nM TRIM30a-specific small ditionally, a neomycin marker cassette flanked by FRT sites and interfering (siRNA) or scrambled siRNA using INTERFERin siRNA a39 loxP site were inserted into the 39 untranslated region of the transfection reagent (Polyplus-Transfection, Illkirch, France) according to LTbR gene. The targeting vector was introduced into mouse ES the manufacturer’s instructions. The siRNA constructs were designed as cells and homologous recombination was verified by Southern described (29). blot and PCR analysis (Fig. 1A, 1B). After germline transmission, TLR tolerance heterozygous LTbR+/flox mice were crossed with FLPe deleter The cells were not stimulated or were preincubated with 10 mg/ml rat IgG, mice to delete the neomycin cassette in vivo. Deletion was veri- 10 mg/ml agonistic anti-mLTbR mAb, or 100 ng/ml LPS (S. enterica se- fied by Southern blot analysis using a neomycin-specific probe rotype abortus equi; Sigma-Aldrich) for 30 h, washed twice with medium, (data not shown). LTbRflox/flox mice were born at expected Men- 3428 LTbR ACTIVATION INDUCES CROSS-TOLERANCE

FIGURE 1. Conditional inactivation of the LTbR gene. (A and B) Generation of LTbR-floxed mice. (A) Southern blot analysis of genomic DNA from E14.1 wild-type and targeted ES cells. Hybridization with the flanking probe yields a 17-kb fragment for the wild-type allele and a 4-kb fragment for the Downloaded from targeted allele. (B) PCR analysis of the integration of the 59 loxP site after successful germline transmission. LTbRflox/wt, tail DNA from heterozygous mice; LTbRflox/flox, tail DNA from homozygous mice; Wt, tail DNA from control mouse. (C–E) Generation of macrophage/neutrophil-specific LTbR-deficient mice (LTbRflox/flox 3 LysM-Cre). (C) Southern blot analysis of LTbR gene deletion in BMDM. (D) Northern blot analysis of LTbR mRNA expression in BMDM derived from the indicated genotypes. (E) FACS analysis verifying the lack of LTbR protein expression on BMDM derived from LTbRflox/flox 3 LysM-Cre compared with the expression on LTbRflox/flox mice. http://www.jimmunol.org/ delian frequencies, appeared healthy, and proofed fertile. To expression after LTbR activation on BMDM derived from wild- generate macrophage- and neutrophil-specific LTbR-deficient type mice revealed that TRIM30a mRNA expression is transiently mice, LTbRflox/flox mice were crossed with LysM-Cre mice (32). induced as early as 8 h after LTbR stimulation, reaching a peak Efficient cell type-specific deletion of the LTbR gene locus was after 16 h stimulation (Fig. 2E). To evaluate TRIM30a pro- verified by Southern blot analysis using BMDM (Fig. 1C). Ex- tein expression in BMDM derived from wild-type, LTbR-defi- pression of LTbR mRNA was efficiently ablated in BMDM of cient mice, LTbRflox/flox 3 LysM-Cre mice, and control mice LTbRflox/flox 3 LysM-Cre mice compared with LTbRflox/flox (LTbRflox/flox) we used a TRIM30a-specific polyclonal antise- control mice (Fig. 1D). Protein expression of the LTbR was rum and analyzed TRIM30a expression after LTbR activation by strongly reduced in BMDM derived from LTbRflox/flox 3 LysM- Western blot analysis. These results clearly demonstrate a tran- by guest on September 27, 2021 Cre mice when compared with BMDM derived from LTbRflox/flox sient LTbR-dependent induction of TRIM30a protein expression mice (Fig. 1E). This result was further substantiated by analyzing consistent with the pattern of its mRNA expression (Fig. 2C, 2D, RPEC and bone marrow-derived neutrophils (BMDN) generated 2F). Interestingly, no TRIM30a protein expression was detected from both genotypes (data not shown). Collectively, these data in BMDM derived from LTbRflox/flox 3 LysM-Cre mice after indicate that LTbR expression was successfully inactivated on LTbR stimulation, supporting the observation of a strongly re- macrophages/neutrophils whereas LTbR expression was not al- duced TRIM30a mRNA induction in these cells (Fig. 2A, 2B). To tered in LTbRflox/flox mice. test for a possible role of neutrophils in our experimental model using LTbRflox/flox 3 LysM-Cre mice, we assessed LTbR ex- b LT R activation on macrophages/neutrophils induces pression on neutrophils (Gr1+, CD11b+, and F4/802) isolated from a TRIM30 expression bone marrow cells (BMDN) derived from both genotypes. Inter- The LTab/LTbR pathway has been defined as being crucial for estingly, only ∼6% of the Gr1+, CD11b+, and F4/802 cell pop- effective host defense (14, 16) and innate immune responses (18, ulation isolated from bone marrow cells (BMDN) derived from 33). However, it is widely unknown which are controlled by LTbRflox/flox mice expressed LTbR on the cell surface. LTbR ex- LTbR signaling in vivo and which are critical for a controlled pression was ablated on neutrophils isolated from LTbRflox/flox 3 innate immune response. This issue was addressed by a compre- LysM-Cre mice (Fig. 2G). Examination of BMDN derived from hensive ex vivo microarray profiling of LTbR-dependent tran- LTbRflox/flox 3 LysM-Cre mice and control mice (LTbRflox/flox) scripts. Using this approach we were able to identify TRIM30a as revealed no significant induction of TRIM30a expression in both a target gene of LTbR signaling (34). genotypes (Fig. 2H). Collectively, these data indicate that LTbR So far, TRIM30a expression has only been shown to negatively expression was successfully inactivated on BMDN derived from regulate TLR-mediated NF-kB activation in BMDCs (29). Indeed, LTbRflox/flox 3 LysM-Cre mice, but no significant TRIM30a in- we found that in addition to LPS, stimulation of BMDM derived duction could be detected after LTbR stimulation in BMDN de- from wild-type mice with an agonistic anti-LTbR mAb induced rived from LTbRflox/flox mice. TRIM30a mRNA expression (Fig. 2A). In contrast, in BMDM Furthermore, we investigated whether LTbR ligands (LTa1b2, derived from LTbR-deficient mice no increase in TRIM30a LIGHT) expressed on activated CD4+ T cells are capable of in- mRNA expression could be detected (Fig. 2A). Examination of ducing TRIM30a expression by activating the LTbR on BMDM. BMDM derived from LTbRflox/flox 3 LysM-Cre mice and control Such a mechanism would require cell–cell contact and imply the mice (LTbRflox/flox) revealed that cell type-specific ablation of possibility of a crosstalk between activated and LTbR expression on BMDM results in a strongly reduced ability macrophages. Cocultivation of different ratios of activated CD4+ to induce TRIM30a expression as a consequence of impaired T cells with BMDM derived from wild-type mice results in an LTbR signaling (Fig. 2B). A time course analysis of TRIM30a increasing induction of TRIM30a mRNA expression (Fig. 3A). In The Journal of Immunology 3429 Downloaded from http://www.jimmunol.org/

FIGURE 2. LTbR signaling induces TRIM30a expression in BMDM. (A and B) Quantitative RT-PCR analysis of TRIM30a induction in BMDM derived from wild-type mice and LTbR-deficient mice (A) or derived from LTbRflox/flox mice and LTbRflox/flox 3 LysM-Cre mice (B) after stimulation with LPS (100 ng/ml), agonistic anti-LTbR mAb (5G11, 10 mg/ml), or rat IgG (10 mg/ml) as control for 16 h. Data are expressed as means 6 SD. Representative data from one out of three independent experiments are shown. (C and D) Western blot analysis of lysates of BMDM derived from wild-type mice and LTbR- by guest on September 27, 2021 deficient mice (C)orLTbRflox/flox mice and LTbRflox/flox 3 LysM-Cre mice (D) after stimulation with LPS (100 ng/ml), agonistic anti-LTbR mAb (5G11, 10 mg/ml), or rat IgG (10 mg/ml) as control for 16 h using a polyclonal anti-TRIM30a or anti–b-actin (loading control) antiserum. (E) Quantitative RT-PCR analysis of TRIM30a expression in BMDM derived from wild-type mice after stimulation with agonistic anti-LTbR mAb (5G11, 10 mg/ml) at indicated time points. Data are expressed as means 6 SD. Representative data from one out of three independent experiments are shown. (F) Western blot analysis of BMDM derived from wild-type mice after stimulation with agonistic anti-LTbR mAb (5G11, 10 mg/ml) at indicated time points using a polyclonal anti- TRIM30a or anti–b-actin (loading control) antiserum. (G) FACS analysis of LTbR protein expression on BMDN derived from LTbRflox/flox 3 LysM-Cre mice compared with the expression on LTbRflox/flox mice. (H) Quantitative RT-PCR analysis of TRIM30a induction in BMDN derived from LTbRflox/flox mice and LTbRflox/flox 3 LysM-Cre mice after stimulation with LPS (100 ng/ml), agonistic anti-LTbR mAb (5G11, 10 mg/ml), or rat IgG (10 mg/ml) as control for 16 h. Data are expressed as means 6 SD. Representative data from one out of two independent experiments are shown. contrast, cocultures of activated T cells with BMDM derived from LTbR activation on macrophages induces tolerance to TLR4 LTbR-deficient mice or cocultures of nonactivated T cells with and TLR9 ligands BMDM derived from wild-type mice did not result in an induction To further confirm that LTbR activation on macrophages results of TRIM30a mRNA expression. Furthermore, no induction of in the induction of TRIM30a expression, we silenced TRIM30a TRIM30a mRNA in BMDM was detected in coculture experi- expression by using siRNA in the mouse macrophage cell line + ments of activated CD4 T cells derived from LTbR ligand- J774, which we found positive for LTbR expression as revealed by + deficient mice or activated CD4 T cells derived from wild-type RT-PCR and FACS analysis. Stimulation of J774 cells with ago- mice after pretreatment with a functional inhibitor of LTbR ac- nistic anti-LTbR mAb in a time-dependent manner resulted in tivation (LTbR:Ig) (Fig. 3A). To test whether LTbR-induced TRIM30a induction to comparable amounts observed when using TRIM30a expression in BMDM is dependent on TNF expres- TLR4 and TLR9 ligands on mRNA as well as on protein level sion and signaling or the activation of the MyD88 signaling (data not shown). Furthermore, we found that TRIM30a mRNA pathway, we generated BMDM from both TNF-deficient and expression after LTbR activation is efficiently inhibited in J774 MyD88-deficient mice and tested TRIM30a induction after cells when transfected with TRIM30a-specific siRNA compared LTbR stimulation (Fig. 3B, 3C). We found that LTbR-mediated with cells transfected with scrambled siRNA used as control (Fig. TRIM30a induction is independent of 1) TNF expression and 4A). We also noted that the expression of endogenous TRIM30a signaling and 2) the MyD88 signaling pathway, indicating a unique protein in nonstimulated J774 cells is inhibited using TRIM30a- LTbR-dependent signaling pathway in BMDM that subsequently specific siRNA (Fig. 4B). More importantly, the induction of induces the expression of TRIM30a. The exact mechanisms by TRIM30a protein expression after LTbR stimulation is efficiently which LTbR activation induces TRIM30a expression in myeloid inhibited using TRIM30a-specific siRNA compared with J774 cells are currently under investigation. cells transfected with scrambled siRNA (Fig. 4B). 3430 LTbR ACTIVATION INDUCES CROSS-TOLERANCE Downloaded from FIGURE 3. LTbR-mediated TRIM30a expression induced by activated T cells is independent of TNF expression and the MyD88 signaling pathway. (A) Quantitative RT-PCR analysis of TRIM30a induction after coculturing of BMDM derived from wild-type mice or LTbR-deficient mice with activated CD4+ T cells isolated from wild-type or LTab/LIGHT double-deficient mice for 24 h. Additionally activated and LTbR:Ig-pretreated CD4+ T cells were incubated with BMDM derived from wild-type mice. Nonactivated CD4+ T cells were used as negative control. Data are expressed as means 6 SD. Representative data from one out of two independent experiments are shown. (B and C) Quantitative RT-PCR analysis of TRIM30a induction in BMDM derived from wild-type mice and MyD88-deficient mice (B) or derived from wild-type mice and TNF-deficient mice (C) after stimulation with LPS (100 ng/ ml), polyinosinic-polycytidylic acid (25 mg/ml) agonistic anti-LTbR mAb (5G11, 10 mg/ml), or rat IgG (10 mg/ml) as control. Data are expressed as http://www.jimmunol.org/ means 6 SD. Representative data from one out of three independent experiments are shown.

Subsequently, we tested whether TRIM30a expression induced mAb (data not shown). Furthermore, we observed a higher cyto- by LTbR activation regulates the production of proinflammatory kine production in nonstimulated J774 cells when expressing mediators. Large amounts of TNF and IL-6 are detectable in TRIM30a-specific siRNA compared with J774 cells expressing the supernatant of J774 cells after stimulation with LPS (TLR4) scrambled siRNA. Again, prestimulation of J774 cells with ago- and CpG (TLR9) whereas stimulation of these cells with ago- nistic anti-LTbR mAb resulted in a significant suppression of TNF

nistic anti-LTbR mAb could not induce proinflammatory cyto- and IL-6 expression when transfected with scrambled siRNA upon by guest on September 27, 2021 kine expression (Fig. 4C, 4D and data not shown). As expected, restimulation with LPS. In contrast, no suppression of TNF and pretreatment of J774 cells with LPS results in a significant sup- IL-6 production was detected in J774 cells prestimulated with pression of TNF and IL-6 expression. Interestingly, prestimula- agonistic anti-LTbR mAb when TRIM30a expression was si- tion of J774 cells with anti-LTbR agonistic mAb resulted in lenced using specific TRIM30a siRNA (Fig. 4C, 4D). These re- a significant suppression of TNF and IL-6 expression compared sults clearly demonstrate that LTbR prestimulation results in the with J774 cells not prestimulated or cells prestimulated with rat suppression of proinflammatory mediators upon TLR restimula- IgG (Fig. 4C, 4D). Additionally, in restimulation experiments tion in a TRIM30a-dependent manner. using LPS and IFN-g, a highly reduced production of NO, a key We next tested whether TRIM30a expression induced by LTbR mediator of the antimicrobial immune response, was observed activation regulates the production of proinflammatory cytokines when J774 cells were prestimulated with agonistic anti-LTbR and mediators in primary macrophages. Large amounts of TNF

FIGURE 4. LTbR signaling inhibits the production of TNF and IL-6 by in- ducing TRIM30a expression. (A and B) Analysis of TRIM30a mRNA expres- sion by quantitative RT-PCR (A)and TRIM30a protein expression by Western blot (B) in J774 cells after transfection (24 h) with TRIM30a-specific siRNA or scrambled siRNA followed by stimu- lation with agonistic anti-LTbRmAb (5G11, 10 mg/ml). (C and D)ELISAof TNF (C)orIL-6(D) using supernatant of J774 cells transfected (24 h) with specific TRIM30a siRNA or scrambled siRNA before stimulated with agonistic anti- LTbR mAb (5G11, 10 mg/ml), rat IgG (10 mg/ml), or LPS (100 ng/ml) for 30 h followed by restimulation with LPS (100 ng/ml) for 8 h. Differences were considered significant with a p value of ,0.05. **p , 0.001, ***p , 0.0001. The Journal of Immunology 3431 and IL-6 are detectable in the supernatant of BMDM derived from duced TNF serum levels compared with wild-type mice pretreated wild-type mice, LTbRflox/flox 3 LysM-Cre mice, or LTbRflox/flox with PBS or rat IgG. Interestingly, pretreatment with agonistic mice after stimulation with TLR4 ligand (LPS) and TLR9 ligand anti-LTbR mAb also resulted in strongly reduced TNF serum (CpG), whereas stimulation with agonistic anti-LTbR mAb alone levels after LPS rechallenge comparable to the levels observed could not induce proinflammatory cytokine expression (Fig. 5A–D after the pretreatment with low doses of LPS. This effect was and data not shown). No difference in the induction of TLR tol- abolished when using inactivated agonistic anti-LTbR mAb, thus erance was observed in BMDM derived from LTbRflox/flox 3 excluding a possible LPS contamination of the Ab preparation LysM-Cre mice or LTbRflox/flox mice using TLR4 and TLR9 (Supplemental Fig. 2E). Furthermore, LTbR-deficient mice were (data not shown). As expected, prestimulation of BMDM no longer able to mount a LPS tolerance induction by the treat- derived from LTbRflox/flox mice but not from LTbRflox/flox 3 ment with anti-LTbR mAb, although reduced TNF serum levels LysM-Cre mice with agonistic anti-LTbR mAb resulted in were detected by pretreatment of these mice with low doses of hyporesponsiveness for the induction of TNF and IL-6 after LPS before LPS rechallenge (Supplemental Fig. 2F). The analysis restimulation with the TLR ligands LPS or CpG (Fig. 5A–D). of LTbRflox/flox 3 LysM-Cre mice in our experimental model These results were further verified by using BMDM derived from demonstrated that pretreatment with agonistic anti-LTbR mAb LTbR-deficient mice and wild-type mice (Supplemental Fig. 2A– does not result in reduced TNF serum levels, suggesting that D). Additionally, restimulation with LPS and IFN-g resulted in LTbR signaling in macrophages seems to be involved in TLR a highly reduced production of NO when using BMDM derived tolerance in our experimental model (Fig 6B). As anticipated, from LTbRflox/flox mice cells prestimulated with agonistic anti- prestimulation of the LTbRinLTbRflox/flox mice resulted in

LTbR mAb in contrast to BMDM derived from LTbRflox/flox 3 strongly reduced TNF serum levels, comparable to levels detected Downloaded from LysM-Cre mice (Fig. 5E). Collectively, these data support the after pretreatment with low doses of LPS (Fig. 6A) or observed conclusion that LTbR activation on macrophages induces after LPS pretreatment of wild-type mice (Supplemental Fig. 2E). TRIM30a expression, which renders these cells hyporesponsive Whereas in both LTbRflox/flox 3 LysM-Cre mice and LTbRflox/flox with respect to proinflammatory cytokine and mediator expression. mice LPS induces TRIM30a expression, the stimulation of LTbRflox/flox mice with agonistic anti-LTbR mAb results in the LTbR activation on macrophages induces TLR4 tolerance upregulation of TRIM30a mRNA expression, and no induction of http://www.jimmunol.org/ in vivo TRIM30a mRNA was detected in LTbRflox/flox 3 LysM-Cre mice To further characterize the function of LTbR-induced TRIM30a (Fig. 6C). This observation matches our additional findings of expression in vivo we made use of the LPS-induced tolerance TRIM30a induction after stimulation with agonistic anti-LTbR model. Pretreatment of wild-type mice with low doses of LPS on mAb in LTbR-deficient mice (Supplemental Fig. 2G). These day 5 and day 3 before LPS rechallenge resulted in strongly re- results clearly demonstrate that LTbR signaling in macrophages by guest on September 27, 2021

FIGURE 5. LTbR activation on BMDM results in the suppression of proinflammatory mediators upon TLR4 and TLR9 restimulation. (A–E)TNF(A, C), IL-6 (B, D), or NO pro- duction (E) in the supernatant of BMDM de- rived from LTbRflox/flox mice and LTbRflox/flox 3 LysM-Cre mice stimulated with agonistic anti- LTbR mAb (5G11, 10 mg/ml) or rat IgG (10 mg/ml) for 30 h before restimulation with LPS (100 ng/ml) (A, B), CpG (1 mM) for 8 h (C, D), or LPS (200 ng/ml) and IFN-g (20 ng/ml) for 24 h (E). Data are expressed as mean 6 SD. Statistical significance was determined using aStudentt test. Differences were considered significant with a p value of ,0.05. **p , 0.001, ***p , 0.0001. Representative data from one out of three independent experiments are shown. 3432 LTbR ACTIVATION INDUCES CROSS-TOLERANCE

FIGURE 6. LTbR-induced TRIM30a ex- pression in BMDM results in cross-tolerance to TLR4 ligands in vivo. (A and B) ELISA of TNF serum levels in LTbRflox/flox mice (n =5) (A)andLTbRflox/flox 3 LysM-Cre mice (n = 5) (B) after pretreatment with agonistic anti- LTbR mAb (5G11, 100 mg/mouse), rat IgG (100 mg/mouse), or LPS (50 mg/kg and 20 mg/kg) and challenge with LPS (10 mg/ mouse) for 1 h. (C) Quantitative RT-PCR analysis of TRIM30a expression in RPEC derived from LTbRflox/flox mice (n =3)and LTbRflox/flox 3 LysM-Cre mice (n =3)after treatment with agonistic anti-LTbRmAb (5G11, 100 mg/mouse), rat IgG (100 mg/ mouse), LPS (50 mg/kg and 20 mg/kg). Data Downloaded from are expressed as means 6 SD. Statistical significance was determined using a Student t test. Differences were considered significant with a p value of ,0.05. *p , 0.05, **p , 0.001. Representative data from one out of two independent experiments are shown. http://www.jimmunol.org/

induces TRIM30a expression, which subsequently results in tol- well as SOCS-1 (36–38). However, these negative regulators erance to the TLR4 ligand in vivo. mediating self and cross-tolerance are induced by TLR signaling

pathways. Based on our observation that activated lymphocytes by guest on September 27, 2021 Discussion expressing LTbR ligands induce TRIM30a expression in myeloid Most studies on LTbR signaling have focused on the organization, cells, it is tempting to speculate that activated lymphocytes as part development, and maintenance of lymphoid tissues and their role of the adaptive immune response interact and communicate with in adaptive immune responses (7). Our data demonstrate that myeloid cells to induce a signaling pathway that negatively reg- LTbR signaling is very important for the control of innate immune ulates the early innate inflammatory response. reactions by inducing a negative feedback mechanism in myeloid TRIM30a is a member of the TRIM superfamily, many of cells while mounting the early innate immune response. LTbR- which are expressed upon TLR activation and in an IFN- mediated induction of TRIM30a seems to be tightly controlled on dependent manner (39, 40). It has been shown that members of mRNA as well as on the protein level, resulting in the suppression the TRIM superfamily are involved in a broad range of biological of proinflammatory cytokine and mediator expression in vitro and processes that are associated with innate immune reactions (41). A in vivo. recent study showed that TRIM30a interacts with TAK1 and In addition to LTbR activation using an agonistic anti-LTbR promotes the degradation of TAB2 and TAB3, resulting in an mAb or activated CD4+ T cells expressing LTbR ligands, we also inhibition of TRAF6 auto-ubiquitylation and consequently the found that recombinant mouse LIGHT was capable of inducing inhibition of NF-kB activation, which ultimately reduced proin- TRIM30a expression in BMDM in vitro (data not shown). So far, flammatory cytokine expression (29). Because TAK1 plays a cru- LIGHT has been described as a costimulatory cytokine in T cell cial role in NF-kB activation through other signaling pathways activation by interacting mainly with HVEM expressed on T cells such as those initiated by TNF and IL-1 (42), TRIM30a might (23, 35), but it is not yet known whether the interaction with the also play a role in regulating inflammatory responses in addition LTbR contributes to TRIM30a induction in vivo. to those initiated by TLR activation. Although activation of TLR-mediated signaling pathways ini- A functional ortholog to TRIM30a in humans has not yet been tiating an early inflammatory response are indispensable for pro- identified based on sequence similarity; however, a similar path- tecting the host against pathogenic organisms, an excessive and/or way seems to operate in human cells. Using the human prolonged activation may lead to both acute and chronic inflam- cell line THP-1 we were able to induce LTbR-mediated TLR matory diseases. Therefore, the intensity and duration of TLR tolerance (data not shown), suggesting that some seem to responses must be tightly regulated. Downregulation of TLR regulate proinflammatory cytokine and mediator expression in an signaling, called TLR tolerance, as well as cross-tolerance among LTbR-dependent manner in human cells of myeloid origin. various TLR ligands might have been developed to prevent ex- The exact molecular mechanisms by which LTbR induces cessive inflammatory damage to the host. Most likely TLR tol- TRIM30a expression in myeloid cells are currently under inves- erance is achieved through multiple mechanisms involving the tigation; however, within this study, using a novel conditional induction of negative regulators such as A20, IRAK-M, MyD88, as LTbR mouse line, important information on the role of LTbR/ The Journal of Immunology 3433

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