Production of IL-8 and TNF-α by internalizing IgG is not mediated by

The TRIM21 intracellular Fc receptor is important in the ADIN process of pathogen recognition involving cytosolic antibody. As an autoantigen, TRIM21 is highly expressed in SLE patients (Ben-Chetrit et al., 1988). We hypothesized that anti-DNA antibodies with high titer in SLE are associated with TRIM21. To investigate whether the cytokine signaling by freely internalized 3D8 IgG is mediated by TRIM21, we purified the 3D8 IgG N434D mutant, which cannot interact with TRIM21 due to a single amino acid change in the CH3 domain. The internalization of the TRIM21 binding mutant was confirmed by confocal microscopy (Fig. 10A). Physical interaction of IgGs (wild-type and N434D) and TRIM21 was confirmed by immunoprecipitation (Fig. 10B). The lysates of THP-1 cells treated with IgGs were precipitated with protein A/G agarose beads. TRIM21 was detected only in the pull-downed lysates from the 3D8 IgG wild type, indicating the interaction between 3D8 IgG wild-type and endogenous TRIM21. When THP-1 cells were treated with 5 µM of IgG proteins for 6 h, the secretion of IL-8 and TNF-α were increased by approximately 40% in the cells treated with 3D8 IgG N434D mutant compared to the cells treated with 3D8 IgG wild-type (Fig. 10C). In addition, IL-8 and TNF-α production by the 3D8 IgG wild-type was increased by 20% in TRIM21-knockdown THP-1 cells (Fig. 10D, E). The results indicate that TRIM21 is not responsible for the production of cytokines by 3D8 IgG, but seems to play a role as a negative regulator, and also suggests the possibility of an unknown intracellular Fc receptor instead of TRIM21.

A B

TRIM21 k chain Fc

Input 10%IP : Protein A/G

TRIM21

Human IgG3D8 IgG wt 3D8 N434D-m

D E

Control TRIM21

0 1000 2000 3000 4000

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Fig. 10. Production of IL-8 and TNF-α by internalizing IgG is not mediated by signaling function of TRIM21. (A) THP-1 cells were treated with 5 µM of the 3D8 IgG-N434D mutant for 6 h at 37°C. After fixation and permeabilization of cells, dylight550 goat α-human IgG Fc was detected by confocal microscopy. (B) Co-immunoprecipitation of 3D8 IgGs and TRIM21. THP-1 cells were treated with 5 µM 3D8 IgGs for 6 h at 37°C. Co-immunoprecipitation with cell lysates was performed using the protein A/G agarose resin. Samples were analyzed by immunoblotting with antibodies specific for TRIM21, Fc, and the κ light chain of human IgG. As input controls, 10% of the total cell lysates were used. (C) THP-1 cells were treated with 5 µM 3D8 IgGs for 6 h at 37°C. The levels of IL-8 and TNF-α in culture supernatants were measured using ELISA. (D) Western blotting assessment of TRIM21 gene knockdown by shRNA in THP-1 cells. (E) The levels of IL-8 and TNF-α were measured by ELISA after 5 µM 3D8 IgG treatment of control and TRIM21-knockdown THP-1 cells for 6 h at 37°C. (C, E) Data are expressed as the mean ± standard error of three independent experiments. All P-values were calculated using a two-tailed Student’s t-test. Statistical significance is indicated on the graphs (**P<0.01; ***P<0.001).

K. 3D8 IgG promotes de-ubiquitination of TRIM21 and negatively regulates IL-8 and TNF-α production.

TRIM21 is an E3 ligase that recognizes the internalized virus-antibody complex in the ADIN mechanism leading to the proteasome-degradation pathway.

Since both antibodies and viruses are degraded by TRIM21, we assumed that TRIM21 leads to degradation by interacting with the Fc region of an antibody, resulting in decreased production of cytokines. Therefore, we examined whether the reduced production of cytokines by treatment with the 3D8 IgG wild-type as compared to 3D8 IgG N434D is related to the proteasomal degradation of 3D8 IgG wild type caused by the ubiquitination activity of TRIM21. Control THP-1 and TRIM21-knockdown cells were incubated with 3D8 IgG wild-type for the indicated times, and then heavy chains of 3D8 IgG were detected in lysate by western blot (Fig.

11A). Heavy chains of 3D8 IgG did not appear to be ubiquitinated or degraded at time points in control cells and TRIM21-knockdown THP-1 cells, indicating that TRIM21 does not lead to degradation of freely internalized IgG.

Based on the finding that the Fc region binding to TRIM21 does not act as a substrate for the E3 ligase and is not degraded, we examined whether auto-ubiquitination of TRIM21 is involved in the mechanism of cytokine production. To compare the mechanism of TRIM21 related to cytokine production, we used THP-1 cells, which secretes IL-8 and TNF-α, and HeLa cells, which do not secrete any cytokines. THP-1 cells and HA-ubiquitin/Flag-TRIM21 overexpressing HeLa cells were incubated with 3D8 IgG and 3D8 IgG N434D for 6 h, and then the lysates were immunoprecipitated with α-ubiquitin and α-HA antibodies, respectively (Fig. 11B, C). In THP-1 cells, ubiquitinated-TRIM21 was reduced by 3D8 IgG compared with control cells, whereas the ubiquitinated-TRIM21 was recovered by treatment of the TRIM21-binding mutant. However, there was no difference in ubiquitination of TRIM21 between the treatment of both two antibodies in HeLa cells.

De-ubiquitination of TRIM21 was observed only in THP-1, suggesting that 3D8 IgG induced de-ubiquitination of TRIM21 in a monocyte-dependent immune response.

3D8 IgG (h)

shTRIM21 shControl

- 0.5 1 2 3 4

hFc

GAPDH TRIM21 - 0.5 1 2 3 4

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B

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- W N - W N

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C

HA-TRIM21 Flag-Ub Protein

IP : Flag

- W N

50 + +

+ +

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- W N

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+ + +

+ +

HeLa

Fig. 11. 3D8 IgG promotes de-ubiquitination of TRIM21, resulting in negative regulation of IL-8 and TNF-α production. (A) Immunoblots for TRIM21 and heavy chains of IgGs in control and TRIM21-knockdown THP-1 cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B, C) Co-immunoprecipitation results. (B) THP-1 cells were incubated with 3D8 IgG wild-type (W) and 3D8 IgG N434D (N) for 6 h at 37°C and detected by western blot. Lysates were immunoprecipitated by α-ubiquitin antibody, and then IP and input sample were detected by α-ubiquitin and α-TRIM21 antibody. (C) HA-TRIM21 and Flag-ubiquitin overexpressed HeLa cells were incubated with 3D8 IgG wild-type and 3D8 IgG N434D for 6 h at 37°C. Lysates were immunoprecipitated using α-Flag antibody, and immunoprecipitated and input samples were detected using α-HA and α-Flag antibody by western blot.

L. IL-8 and TNF-α responses to internalizing IgG are produced by NF-κB activation and MAPK-signaling.

Gene expression of IL-8 is regulated by the activation of NF-κB and AP-1 transcription factors through the pathways involving the mitogen-activated protein kinase (MAPK) family of proteins including c-Jun, N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). With this knowledge, we sought to determine if the TAK1-linked canonical NF-κB signaling pathway and MAPKs are activated by 3D8 IgG with or without the pre-treatment of specific inhibitors (Fig.

12A). The inhibitors were 5z-7-oxozeaenol (TAK1 inhibitor), IKK inhibitor VII (IKKα/β inhibitor), SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), and SP600125 (JNK inhibitor). These inhibitors significantly reduced the production of IL-8 and TNF-α by 3D8 IgG in THP-1. IL-8 production was nearly abrogated (approximately 99% reduction) by 5z-7-oxozeaenol, and by 66% using IKK inhibitor VII, 53% using SB202190, 73% using PD98059, and 81% using SP600125 compared with 3D8 IgG alone. TNF-α production was also dramatically reduced by approximately 92% using 5z-7-oxozeaenol, 63% using IKK inhibitor, 62% using SB202190, 82% using PD98059, and 94% using SP600125 compared with 3D8 IgG alone. The role of NF-κB in the cytokine production by 3D8 IgG was confirmed by the observation that pre-treatment of cells with 5z-7-oxozeaenol or IKK inhibitor inhibited NF-κB luciferase reporter activity (Fig. 12B). Moreover, 10 min after treatment with 3D8 IgG, phosphorylation of MAPKs was observed by western blot, compared to treatment with human IgG (Fig. 12C). The results indicated that TAK1 is an upstream signaling molecule capable of activating NF-κB signaling and MAPKs (p38, ERK, and JNK).

A

Fig. 12. IL-8 and TNF-α responses to internalizing IgG are produced by NF-κB activation and MAPK-signaling. (A) Effect of pharmacological inhibitors on cytokine production by 3D8 IgG treatment of THP-1 cells as determined by ELISA.

(B) Luciferase assay for NF-kB promoter activation. NF-κB reporter THP-1 cells were pre-treated with 5z-7-oxozeaenol for 1 h and then incubated with 5 µM 3D8 IgG for 24 h. Cells were harvested and luciferase assays were performed. (**P<0.01;

***P<0.001 versus treatment with 3D8 IgG alone). (C) 3D8 IgG and human IgG (5 µM) were incubated with THP-1 cells for the indicated times and the lysates were examined using antibody to MAPKs (p38, ERK, and JNK) and phospho-MAPKs by western blot.

C

Ⅳ. Discussion

Anti-DNA antibody that is present in the blood is a hallmark of SLE. The antibody causes inflammation when deposited in tissues, such as the glomeruli (Holvoet et al., 1989). However, how the deposition occurs and how it triggers inflammation are unclear. It is important to elucidate the pathological mechanism associated with anti-DNA antibodies

The pathogenesis that has been studied in relation to anti-DNA antibodies is the inflammatory response caused by the production of IFN-β by the internalization of DNA/anti-DNA immune complex (DNA-IC) in pDCs. DNA-ICs are phagocytosed via the Fcγ receptor and are recognized by TLR9 to produce IFN-β, which promotes an inflammatory response (Bave et al., 2003; Henault et al., 2012;

Henault et al., 2016). Internalized DNA-IC recognized by TLR9 produces IL-1β as a result of NLRP3 activation in monocytes and macrophages (Shin et al., 2013).

In this study, the internalizing anti-DNA antibody, 3D8 IgG, elicited immune signaling without forming immune complexes. Production of IL-8 and TNF-α was observed only in human monocytes among other immune and non-immune cells (Fig. 1). 3D8 IgG induced secretion of IL-8 and TNF-α via an alternative novel pathway for Fc-mediated cytokine signaling. The pathway was distinct from the pathway mediated by TRIM21, which is an intracellular Fc receptor (Fig. 10). Moreover, unlike the induction of an inflammatory response by DNA-ICs, 3D8 IgG was not recognized by TLR9 (Fig. 5). The findings suggest the presence of unknown Fc receptors, recognizing cytosolic antibody. The data provides novel insight into the pathogenesis of SLE.

The pathogenesis of SLE by anti-DNA antibodies has been reported in relation to the characteristics of variable domains of antibodies because anti-DNA antibody is polyreactive with autoantigens (Oelke and Richardson, 2002; Giles and Boackle, 2013; Rekvig and Van der Vlag, 2014). The antibody binds to DNA and

also to several autoantigens, including phosphoglycerate kinase-1 (PGK-1), annexin II, and TLR4 to stimulate the production of pro-inflammatory cytokines, endoplasmic reticulum stress, and apoptosis. These cellular events are common features that begin with the variable region of the antibody, promoting overproduction of cytokine that has been implicated in the autoantigen-mediated pathogenesis of SLE. Presently, only two 3D8 formats, 3D8 IgG and 3D8 scFv-Fc, induced inflammatory cytokine signaling, even though 3D8 scFv was also internalized into the cytosol of cells (Fig. 3). Our results demonstrate that free-internalized anti-DNA antibody can be mediated by the Fc region and recognized by the intracellular Fc receptor, although the internalizing capacity of the IgG is determined by the variable region (Jang et al., 2009; Pham et al., 2012).

TRIM21 is an intracellular Fc receptor that is a key molecule in the ADIN process. When an antibody-virus complex enters the cytosol of cells, it is recognized by TRIM21, leading to activation of the proteasome-degradation pathway that neutralizes the virus and promotes the secretion of pro-inflammatory cytokines (Mallery et al., 2010; McEwan et al., 2013). In the TRIM21-mediated immune response, the recognition of an antigen-binding antibody initiates various immune responses that include production of cytokines and protein degradation. The 9D11-Tat antibody targets HBx by the fusion of a cell penetrating peptide, TAT, on the C-terminus of the heavy chain and is internalized into the cell cytosol. 9D11-TAT bound to HBx is recognized by TRIM21, which stimulates the activation of NF-κB, AP-1, and IFN-β via the TRIM21-mediated pathway (Zhang et al., 2018). When an antibody targeting the intracellular proteins is microinjected, TRIM21 recognizes the infected antibody, leading to proteasome-mediated degradation of target proteins (Clift et al., 2017). In addition, transfection of antibody-coated latex beads results in TRIM21-induced activation of NF-κB, but transfection of antibodies or beads alone does not (McEwan et al., 2013). The findings imply that the minimum requirement for TRIM21-mediated immune signaling is the presence of IgG antibody in which

antigen-binding sites are occupied by insoluble large antigens, irrespective of living pathogens or even inanimate objects like latex beads in the cytosol. 3D8 IgG is internalized into the cytosol in the absence of antigen where it is recognized by TRIM21. Therefore, it is likely that IgG antibody of the internalized ICs induces TRIM21-mediated immune signaling, whereas the free form of antibody internalized to the cytosol does not, even if IgG is recognized by TRIM21. When the antigen occupies the antigen-binding site, a conformational change of the Fc domain occurs (Sagawa et al., 2005). TRIM21 appears to discriminate against structural changes due to antigen binding.

TRIM21 also interacts with the p97/VCP. In the endoplasmic reticulum-associated degradation (ERAD) system, p97/VCP play a role in the degradation of unfolded IgG. Unfolded IgG is transported from the endoplasmic reticulum to the cytosol of cells where it interacts with VCP. TRIM21 ubiquitinates IgG Fc, which results in antibody degradation by the ERAD system (Takahata et al., 2008). We hypothesized that the inhibition of cytokine production by treatment of 3D8 IgG wild-type by TRIM21 compared to treatment with 3D8 IgG N434D is due to the binding of TRIM21 to 3D8 IgG wild-type, with the induction of proteasomal degradation and decreased intracellular levels. However, presently no intracellular level and molecular mass of 3D8 IgG1 heavy chain were observed for 4 h following treatment in THP-1 cells, implying that the cellular level of the internalized free IgG molecules is not regulated by E3 ligase activity of TRIM21 (Fig. 11A). Stimulation of TRIM21 by the internalized free IgG may differ from the stimulation by unfolded IgG and IgG participating in IC formation. Thus, it is possible that that TRIM21 acts as a negative regulator of the signal transduction pathway initiated by the intracellular Fc sensor, rather than directly regulating the antibody.

TRIM21 is not a key molecule in the immune response in response to 3D8 IgG, but it seems to modulate the production of cytokines by removing ubiquitin from itself. 3D8 IgG binds to an as-yet unknown intracellular Fc receptor to initiate

immune signaling for production of cytokines and also binds to TRIM21. At that time, de-ubiquitin enzymes, such as POH1 or USP4, might recognize TRIM21-IgG complex and catalyze the removal of ubiquitin from TRIM21 (Wada et al., 2006;

Fletcher et al., 2015). In contrast, 3D8 IgG N434D activates immune response by interacting with the Fc receptor but does not remove the ubiquitin because de-ubiquitin enzymes function by recognizing the complex of TRIM21 and IgG. As a result, the signaling pathways for activation of NF-κB and MAPKs are inhibited. The E3 ligase activity of TRIM21 regulates a series of immune mechanisms by ubiquitination to the substrate or itself. This study provides the first evidence that the de-ubiquitination of TRIM21 regulates the immune signaling cascade.

The ability of anti-DNA antibodies to enter cells has been amply described.

However, the proportion of internalizing anti-DNA antibodies present in the serum of SLE patients related to disease activity has not been determined experimentally and there is no quantitative analysis. A correlation between the level of internalizing anti-DNA antibody present in the serum and inflammatory cytokine production may reveal a potential role for the internalization of anti-DNA antibodies in the SLE.

The collective data highlights the importance in identifying the intracellular antibody receptor and the pathway of inflammation. The results support a new mechanism of SLE pathogenesis via the Fc region of an antibody and the proposed pathway is summarized in Fig. 13. Further studies should focus on finding the unknown Fc receptor that initiates the production of the cytokines and elucidating the de-ubiquitination mechanism of TRIM21. Many efforts have been made to develop antibodies targeting intracellular proteins, such as RT11 iMab in the development of therapeutic antibodies (Shin et al., 2017). Our results suggest that the inflammatory response by TRIM21 should be considered in the development of antibodies targeting intracellular proteins. The presence of antibodies in the cytoplasm is not a usual situation. Therefore, attempts to introduce therapeutic antibodies into cells can be perceived by cells as a danger signal. Although our study

was limited to monocytes, the broader suggestion is that antibodies present in the cytoplasm induce an inflammatory response in an Fc region-dependent manner.

Therefore, when an antibody targeting intracellular molecules is delivered into the cytosol of cells, production of pro-inflammatory cytokine must be considered.

Fig. 13. The proposed inflammation mechanism by internalizing anti-DNA antibody in human monocyte.

A novel intracellular Fc sensor ?

Inflammatory cytokines (IL-8,TNF-a) Cytosol

TRIM21 3D8 IgG wild type

3D8 IgG N434D mutant

TAK1

MAPKs IKK

NF-kB

Human monocyte Nucleus

(−)

HSPGs CSPGs

Ubiquitin

Ⅴ. Conclusion

We conclude that the internalizing anti-DNA antibody, 3D8 IgG, promotes the production of cytokines (IL-8 and TNF-α), activation of MAPKs (p38, ERK, and JNK), and activation of NF-κB transcription factor in Fc-dependent manner, but does not depend on DNA-binding activity by the variable region in human monocytes.

Unlike the mechanism of inflammation by the DNA/anti-DNA IC, which is an established pathogenic mechanism of SLE, 3D8 IgG is internalized into cells via clathrin-mediated endocytosis mediated by HSPG and CSPG on the cell surface. The TRIM21 intracellular Fc receptor negatively regulates the Fc-mediated immune response of 3D8 IgG. 3D8 IgG inhibits the immune response initiated by an unknown intracellular Fc receptor by removing the auto-ubiquitin of TRIM21. Our results demonstrate the existence of a potent cellular mechanism that allows human monocytes to stimulate pro-inflammatory cytokine production by detecting the free form of the IgG molecule in the cytosol via the Fc-dependent pathway.

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