Multifaceted roles of TRIM38 in innate immune and inflammatory responses

Abstract

The tripartite motif-containing (TRIM) proteins represent the largest E3 ubiquitin ligase family. The multifaceted roles of TRIM38 in innate immunity and inflammation have been intensively investigated in recent years. TRIM38 is essential for cytosolic RNA or DNA sensor-mediated innate immune responses to both RNA and DNA viruses, while negatively regulating TLR3/4- and TNF/IL-1β-triggered inflammatory responses. In these processes, TRIM38 acts as an E3 ubiquitin or SUMO ligase, which targets key cellular signaling components, or as an enzymatic activity-independent regulator. This review summarizes recent advances that highlight the critical roles of TRIM38 in the regulation of proper innate immune and inflammatory responses.

INTRODUCTION

The innate immune system is the first line of host defense against infection of microbial pathogens. Host cells express several types of germline-encoded pattern-recognition receptors (PRRs), which sense a wide range of pathogenic components that are named pathogen-associated molecular patterns (PAMPs), including nucleic acids, lipids, proteins and so on.¹ According to their subcellular locations and structures, PRRs can be grouped in several families, including plasma or endosomal membrane-bound Toll-like receptors (TLRs), cytosolic RIG-I-like receptors (RLRs), cytosolic DNA sensors and cytosolic NOD-like receptors (NLRs).^{2, 3, 4, 5} The membrane-bound TLRs are mainly expressed in immune cells, while RLRs and DNA sensors recognize RNA or DNA in a variety of cell types, including both immune and non-immune cells.⁶ The NLR family contains more than 20 members. Several members of this family form inflammasomes and trigger inflammatory responses, including the secretion of interleukin (IL)-1β via the activation of caspase-1, in response to various pathogenic stimulations.^{7, 8} The signaling through TLRs, RLRs, DNA sensors and NLRs culminates in the expression of downstream host defense genes, such as type I interferons (IFNs) and inflammatory cytokines, to inhibit replication of pathogens, clear pathogen-infected cells, and facilitate adaptive immune response.⁹

Various mechanisms regulate innate immune and inflammatory responses. In the past years, increasing evidence suggests that members of the tripartite motif (TRIM) family, which is the largest family of RING domain-containing E3 ligases, have critical regulatory roles in innate immunity and inflammation.^{10, 11} Although most TRIM family members are E3 ubiquitin ligases, some members of the TRIM family have been suggested to confer E3 ligase activity for ubiquitin-like modifiers (UbLs), such as SUMO, NEDD8 and ISG15.^{12, 13, 14, 15, 16, 17} Among the TRIM family members, TRIM38 has been demonstrated to have important regulatory roles through distinct mechanisms in various innate immune and inflammatory pathways, which are the focus of this review.

STRUCTURE OF TRIM38

The TRIM proteins derive their names from their common N-terminal tripartite RBCC motif, which consists of a RING domain, one or two BBox domains and a coiled-coil domain (CCD).¹¹ The BBox exhibits zinc-finger structure that is highly similar to the RING domain.^{18, 19} Because of the high similarity of BBox to RING domain, it has been suggested that BBox offers an E2 binding site similar to RING and thereby confers E3 ligase activity to some TRIM proteins lacking a RING domain. For example, a recent work has demonstrated that TRIM16, a TRIM protein lacking a RING domain, confers E3 ubiquitin ligase activity in vitro.²⁰ The CCD is necessary and sufficient for oligomerization of TRIM proteins.^{21, 22, 23} In addition, systematic studies have demonstrated that TRIM hetero-oligomers are formed at least in vitro, which increases the spectrum of their biological functions.^{20, 24} The C-terminal domains found in TRIM proteins are quite diverse. The most universal C-terminal domain is PRY-SPRY (B30.2), which is present in most TRIM proteins, including TRIM38. The reported functions of the PRY-SPRY domain are divergent. Predominantly, this domain mediates protein-protein interactions,²⁵ which entails the binding of ubiquitination substrates and determining E3 ligase specificity. In addition, the PRY-SPRY domain is critical for the direct antiviral restriction activity of certain TRIM proteins such as TRIM5.²⁶ TRIM38 is a typical TRIM protein and contains a RING, two BBoxes, a CCD and a PRY-SPRY domain.²⁷ It has been shown that amino acids C16 and C31 in the RING are critical for the optimal catalytic activity of TRIM38, and mutation of either of these cysteines severely impairs TRIM38-mediated polyubiquitination of its substrates.^{28, 29, 30, 31}

INDUCIBLE EXPRESSION OF TRIM38

TRIM38 is ubiquitously expressed in different cell types, such as various human and murine cell lines (HEK293, HeLa, HCT116, A549, THP-1, and RAW264.7), mouse lung fibroblasts (MLFs), bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs).^{27, 28, 29, 30, 31} Expression of TRIM38 can be induced by various stimuli, such as TLR ligands, type I IFNs, and viral infection, suggesting that TRIM38 is a potential interferon-stimulating gene (ISG).^{28, 29, 30}

NEGATIVE REGULATION OF TRIM38 on TLR-MEDIATED SIGNALING PATHWAYS

TLRs recognize a set of pathogenic components and have critical roles in host defenses against certain microbes. So far, 10 TLRs have been reported in humans (TLR1–10), while there are 12 known TLRs in mice (TLR1-9 and TLR11–13).^{2, 32} TLRs contain an extracellular domain to which ligands bind, a transmembrane domain, and a conserved cytoplasmic Toll/IL-1R (TIR) domain, which acts as a platform for the recruitment of downstream TIR domain-containing adapter proteins and other signaling components upon ligand stimulation.² Among the TLRs, TLR3 recognizes viral dsRNA, as well as its synthetic analog polyinosinic-polycytidylic acid [poly(I:C)] in the endosomes.³³ Upon activation, TLR3 recruits a co-receptor MEX3B, an accessory protein WDFY1, and the critical adapter TIR domain-containing adapter TRIF (also called TICAM-1).^{2, 34, 35} TRIF, in turn, recruits TRAF2/6 and two kinase complexes: the IKK complex to activate NF-κB and the TBK1 complex to activate IRF3, leading to subsequent induction of proinflammatory cytokines, such as TNF and IL-1β, type I IFNs and ISGs.¹ Most other TLRs trigger signaling through the MyD88-TRAF6-IKK axis to activate NF-κB but not IRF3.¹ TLR4, which recognizes lipopolysaccharides (LPS) of gram-negative bacteria, is the only receptor that signals through MyD88-dependent pathways to activate NF-κB and TRIF-dependent pathways to activate both NF-κB and IRF3.³⁶

In mouse RAW264.7 cells, it has been demonstrated that Trim38 (referred to as the mouse ortholog of human TRIM38) negatively regulates TLR3/4-mediated NF-κB activation by targeting TRAF6 for proteasomal degradation.³⁰ Furthermore, Trim38 also targets NAP1 for proteasomal degradation, which leads to negative regulation of TLR3/4-mediated IRF3 activation and type I IFN induction.²⁹ An independent study demonstrates that TRIM38 negatively regulates TLR3-mediated activation of IRF3 and induction of type I IFNs by mediating proteasomal degradation of TRIF in human cell lines,³¹ which represents a distinct mechanism from the previous report. Mouse gene knockout studies suggest that Trim38-deficiency potentiates poly(I:C)- and LPS-induced, but not R848 (a ligand for TLR7)- or PGN (a ligand for TLR2)-induced expression of type I IFNs and proinflammatory cytokines in BMDMs, BMDCs and MLFs.²⁸ Trim38-deficiency also increases the serum cytokine levels induced by poly(I:C) and LPS, as well as susceptibility to body weight loss and death triggered by administration of poly(I:C) or LPS or infection with S. typhimurium.²⁸ Biochemical experiments indicate that Trim38-deficiency abolishes K48-linked polyubiquitination of Trif and markedly upregulates the protein level of Trif, suggesting that Trim38 targets Trif for K48-linked polyubiquitination and degradation.²⁸ The mechanisms responsible for the negative regulatory roles of TRIM38 on TLR3/4-mediated signaling are illustrated in Figure 1. Previously, it has been reported that another E3 ubiquitin ligase, WWP2, also targets TRIF for K48-linked polyubiquitination and proteasomal degradation.³⁷ However, WWP2 functions differently with TRIM38. WWP2 specifically regulates TLR3- but not TLR4-mediated innate immune and inflammatory responses. Furthermore, WWP2-deficiency increases TLR3-mediated induction of cytokines in BMDMs but not in BMDCs, whereas Trim38-deficiency increases TLR3/4-mediated induction of cytokines in both cell types. It is possible that Trim38 and WWP2 function in different cell types and distinct pathways.

Figure 1.

TRIM38-mediated negative regulation of TLR3/4-mediated and TNF/IL-1-triggered signaling. After the activation of TLR3/4, TRIM38 is recruited to the adapter protein TRIF, leading to its K48-linked polyubiquitination and degradation, therefore negatively regulating TLR3/4-mediated induction of proinflammatory cytokines and type I IFNs. In the early phase of infection, type I IFNs induce the expression of TRIM38, which in turn mediates the degradation of TAB2/3 by a lysosomal pathway, leading to negative regulation of TNF- and IL-1-triggered signaling and inflammatory response.

POSITIVE REGULATION OF RLR-MEDIATED INNATE IMMUNE RESPONSE BY TRIM38

RLRs, including RIG-I and MDA5, contain two N-terminal tandem CARD domains, an RNA helicase domain and a C-terminal domain (CTD), and recognize RNAs of different RNA viruses.³⁸ In the absence of viral infection, RIG-I and MDA5 are phosphorylated in their respective CARDs to suppress their activation in resting cells.^{39, 40, 41} After recognition of cytosolic viral RNA, RIG-I and MDA5 undergo conformational changes and recruit PP1 for their dephosphorylation,⁴¹ leading to their further recruitment of several E3 ubiquitin ligases, including TRIM25, RNF135 and TRIM4 for K63-linked polyubiquitination,^{42, 43, 44} followed by their translocation to the outer membrane of mitochondria, where they activate the central adapter VISA (MAVS, CARDIF and IPS-1).^{45, 46, 47, 48} VISA, together with WDR5 and TRIM14, in turn recruits TRAF3, the TANK-TBK1-NAP1 complex and the transcriptional factor IRF3.^{49, 50} A recent study has indicated that MSX1 is critical for optimal assembly of the TANK-TBK1-NAP1 complex.⁵¹ Furthermore, in this process, GSK3β is recruited to TBK1 and promotes the self-association and trans-phosphorylation of TBK1, followed by TBK1-mediated phosphorylation of IRF3, leading to the dimerization and translocation of IRF3 to the nucleus.⁵² VISA also recruits TRAF2/6 and the IKK complex, which then phosphorylate IκBα and activate the transcriptional factor NF-κB, leading to translocation of NF-κB to the nucleus. The translocated IRF3 and NF-κB cooperatively drive the transcription of type I IFN genes.^{53, 54} In the late phase of viral infection, RIG-I and MDA5 as well as VISA are regulated by K48-linked polyubiquitination and degradation to avoid their sustained activation.^{55, 56, 57, 58, 59, 60, 61}

In addition to polyubiquitination and phosphorylation, sumoylation of RIG-I and MDA5 has also been reported.^{62, 63} Our recent study suggests that TRIM38 is associated with MDA5 and RIG-I and positively regulates MDA5- and RIG-I-mediated induction of downstream antiviral genes.⁶⁴ Gene knockout in mice suggests that Trim38 is essential for efficient induction of type I IFNs, proinflammatory cytokines and other downstream antiviral genes as well as for host defense against RNA viruses in vivo.⁶⁴ Biochemical analysis suggests that Trim38 acts as an E3 SUMO1 ligase for Mda5 (referred to as the mouse ortholog of human MDA5) and Rig-I (referred to as the mouse ortholog of human RIG-I). Trim38 catalyzes the sumoylation of Mda5 at K43/K865 and Rig-i at K96/K889. In uninfected cells, K43 of Mda5 is basally sumoylated. Upon viral infection, the sumoylation at K43 is enhanced, and K865 is further sumoylated. Similarly, K889 of Rig-i is basally sumoylated in uninfected cells, and K96 is further sumoylated upon viral infection. Trim38-mediated sumoylations of Mda5 and Rig-i are important for antagonizing their K48-linked polyubiquitination and degradation in uninfected and early-infected cells. Previously, it has been demonstrated that dephosphorylation of Mda5 at S88 and Rig-I at S8/T177 by the phosphatase PP1 following viral infection is critical for their activation.⁴¹ Mutagenesis suggests that sumoylation of Mda5 at K43 and Rig-i at K96 is essential for their dephosphorylation and activation following viral infection.⁶⁴ These studies provide solid evidence that sumoylation of Mda5 and Rig-i is essential for the efficient onset of innate immune response to RNA viruses. In addition, these findings suggest that Mda5 and Rig-i are regulated by Trim38-mediated sumoylation with similar mechanisms. Interestingly, it is reported that prolonged EV71 infection causes degradation of TRIM38 in human cells, implying that inactivation of TRIM38 following viral infection is an important immune evasion strategy of RNA viruses.⁶⁵

POSITIVE REGULATION OF THE cGAS-MITA/STING PATHWAYS BY TRIM38

Cytosolic DNA derived from viruses, bacteria and the damaged host cells induces innate immune responses.⁶⁶ Although several DNA sensors have been reported to recognize various DNAs in different cell types, it is widely believed that the cyclic GMP-AMP (cGAMP) synthase (cGAS) is the major sensor of cytosolic DNA in divergent cell types.^{67, 68, 69, 70, 71, 72, 73, 74, 75, 76}

After binding of cytosolic viral or cellular DNA, cGAS undergoes oligomerization and utilizes ATP and GTP for the synthesis of cyclic GMP–AMP (cGAMP), which then acts as a second messenger to bind to and activate the central adapter protein MITA (also called STING).^{77, 78, 79, 80} MITA/STING is translocated from the ER to ER-Golgi intermediate compartments (ERGIC) and the Golgi apparatus.⁸¹ In this process, it has been shown that the ER-associated protein ZDHHC1 facilitates the oligomerization and optimal activation of MITA/STING.⁸² In addition, another ER-associated protein, called iRhom2, facilitates translocation and stability of MITA/STING.⁸³ At ERGIC/Golgi apparatus, the kinases TBK1 and IKK are recruited to the MITA/STING-associated complex in which they phosphorylate IRF3 and IκBα, respectively, leading to the induction of downstream antiviral genes.⁸⁴ In the late phase of viral infection, MITA/STING is further translocated to perinuclear microsomes, in which it is degraded via a lysosome-dependent pathway.^{81, 85}

Post-translational modifications, including phosphorylation, polyubiquitination and glutamylation, have important roles in regulating the cGAS-MITA/STING pathways.^{9, 86, 87, 88} For example, TTLL4/6 catalyzes the glutamylation of cGAS, which impairs its DNA-binding and synthase activity in resting cells. Upon viral infection, the glutamylation modification of cGAS is removed by CCP5/6, leading to the activation of cGAS.⁸⁷ AKT1 phosphorylates cGAS at K305, which inhibits DNA binding to cGAS and avoids its sustained activation.⁸⁹ MITA/STING is also regulated by phosphorylation. ULK1 phosphorylates MITA/STING at S366 upon DNA or cGAMP stimulation, leading to attenuated IRF3 activation.⁸⁵ TBK1 has been shown to phosphorylate MITA/STING at the same residue but positively regulates MITA/STING-mediated signaling.⁹⁰ In addition to phosphorylation, MITA/STING is also modified by various types of polyubiquitin chains, which distinctly regulate the activity of MITA/STING. TRIM56 and TRIM32 catalyze K63-linked polyubiquitination of MITA/STING, leading to its activation.^{91, 92} AFMR catalyzes K27-linked polyubiquitination of MITA/STING, which forms a platform for TBK1 recruitment.⁹³ RNF5 catalyzes K48-linked polyubiquitination of MITA/STING, causing the proteasomal degradation of MITA/STING.⁹⁴ RNF26 catalyzes K11-linked polyubiquitination of MITA/STING, which unlocks its K48-linked polyubiquitination and prevents its proteasomal degradation. RNF26 also appears to negatively regulate innate immune signaling in a temporal fashion.⁹⁵

Recently, it has been demonstrated that TRIM38 is a SUMO ligase for both cGAS and MITA/STING.⁹⁶ Sumoylation of cGAS and STING kinetically regulates the innate immune response to DNA viral infection (Figure 2). In uninfected cells, Trim38 catalyzes sumoylation of cGas at K217, which antagonizes its K48-linked polyubiquitination at K271 and degradation by the ubiquitin-proteasomal pathway. Upon viral infection, Trim38 further catalyzes the sumoylation of cGas at K464, which prevents it from K48-linked polyubiquitination at the same residue and degradation by the ubiquitin-proteasomal pathway. These processes ensure the proper level of cGas and its activation in the early phase of viral infection. The activated cGas catalyzes the synthesis of cGAMP, which binds to and activates the ER-associated Mita/Sting. In the early phase of viral infection, Trim38 also targets Mita/Sting for sumoylation at K337, which promotes its CTT-mediated oligomerization and prevents its degradation by the CMA pathway. These actions result in optimal activation of Mita/Sting as well as induction of downstream antiviral genes. In the late phase of infection, cGas and Mita/Sting are desumoylated by SENP2, leading to their K48-linked polyubiquitination-proteasomal and CMA degradation, respectively. Therefore, the temporal sumoylation of cGas/Mita/Sting by Trim38 and their desumoylation by Senp2 provide important regulatory mechanisms for efficient innate antiviral response at the early phase of infection and its timely termination at the late phase of infection.

Figure 2.

Sumoylation promotes the stability of cGas and Sting and regulates the kinetics of the response to a DNA virus. In uninfected or early-infected cells, cGas and Sting are sumoylated, which promotes their stability and activation by inhibiting K48-linked polyubiquitin-proteasomal and chaperone-mediated degradation (CMA) pathways, respectively, thus promoting an efficient innate immune response to a DNA virus. In the late phase of viral infection, Senp2 mediates the desumoylation of cGas and Sting, leading to their degradation by the proteasomal pathway and CMA, respectively, therefore turning off the innate immune response.

REGULATION OF THE TNF/IL-1β-TRIGGERED INFLAMMATORY RESPONSE BY TRIM38

The proinflammatory cytokines TNF and IL-1β have central roles in many diseases, such as inflammation, autoimmunity and cancers. After the binding of TNF to TNF receptor 1 (TNFR1), the receptor recruits TRADD, TRAF2/5, cIAP1/2 and RIP1 to form a large receptor-associated complex, in which RIP1 undergoes K63-linked polyubiquitination. The TAK1-associated chaperones TAB2 and TAB3 bind to K63-linked polyubiquitin chains of RIP1, which in turn activates downstream kinases, leading to the activation of transcription factors NF-κB and AP1.^{97, 98}

IL-1β binds to IL-1 receptor (IL-1R), leading to recruitment of the IL-1R accessory protein (IL-1RAcP) and the adapter protein MyD88. MyD88 further recruits IRAK1/4 and TRAF6 to the receptor complex, in which TRAF6 catalyzes K63-linked autoubiquitination and/or the synthesis of unanchored K63-linked polyubiquitin chains. These polyubiquitin chains recruit the TAK1-TAB1-TAB2/3 complex, causing TAK1 autophosphorylation and activation. TAK1 ultimately activates NF-κB and MAPKs, leading to the induction of various proinflammatory cytokines and chemokines.^{97, 99}

TNF- and IL-1β-triggered signaling is timely downregulated or terminated to avoid excessive inflammatory responses in host cells. Several proteins have been reported to terminate TNF and IL-1β signaling by targeting various signaling components in the pathways. For example, MARCH8, cIAP1/2 and RBCK1 have been shown to induce K48-linked polyubiquitination and proteasome-dependent degradation of ILRAcP, RIP1 and TAB2/3, respectively.^{100, 101, 102} TRIM5α interacts with the TAK1-TAB1-TAB2/3 complex and promotes TAB2 degradation via a lysosome-dependent approach.¹⁰³ Several deubiquitinating enzymes have also been shown to have negative regulatory roles in TNF and IL-1β signaling. A20, USP2a, USP4 and USP20 mediate deubiquitination of TRAF6,^{104, 105, 106, 107} whereas CYLD deubiquitinates TRAF6 and IKKγ.^{108, 109} In addition, A20, CYLD, and USP4 mediate deubiquitination of RIP1, TRAF2 and TAK1, respectively.^{108, 110, 111} It has also been shown that DUSP14 catalyzes dephosphorylation of TAK1, leading to suppression of TNF and IL-1β-triggered signaling.¹¹² It is possible that different enzymes target distinct signaling components in various cell types following TNF or IL-1β stimulation.

Recently, it has been shown that TRIM38 negatively regulates TNF- and IL-1β-triggered activation of NF-κB and MAPKs as well as inflammatory responses.²⁷ TRIM38 promotes the degradation of TAB2/3 through a lysosomal-dependent pathway, which requires its C-terminal PRY-SPRY but not the RING domain.²⁷ The degradation of TAB2/3 inhibits recruitment of TAK1 to upstream adapters RIP1 and TRAF6, leading to inhibition of NF-κB and MAPKs and the expression of inflammatory cytokines.²⁷ TRIM38 is highly induced by type I IFNs and negatively regulates TNF/IL-1β signaling in IFN-β-primed but not unprimed mouse immune cells.²⁸ These findings suggest that TRIM38 probably has important negative regulatory roles in the late phase of inflammatory response to various pathogenic stimuli, which trigger induction of type I IFNs at the early phase of infection (Figure 1).

TRIM38 IN AUTOIMMUNE DISEASES

Several TRIM family proteins have been reported to be associated with certain autoimmune diseases.^{113, 114} A recent study clearly demonstrates that TRIM38 is a valid target for auto-antibodies in primary Sjögren's syndrome (SS).¹¹⁵ In primary SS, the presence of anti-TRIM21 has been associated with increased disease severity.¹¹⁶ It has also been shown that anti-TRIM38 positivity is significantly associated with the presence of auto-antibodies to TRIM21. However, how TRIM38 is involved in autoimmune diseases is still unknown. It has been shown that mouse TRIM21-reactive antibodies penetrate live salivary gland cells in a mouse model.¹¹⁷ It is possible that anti-TRIM38 penetrates live cells, causing dysregulated inflammatory responses.

Several studies suggest that constitutive activation of MDA5 and RIG-I derived from viral infection or their genetic mutations is associated with three different types of autoimmune diseases including Aicardi-Goutières syndrome, systemic lupus erythematosus and Singleton–Merten syndrome.^{118, 119, 120, 121, 122} Deregulation of the cGAS-MITA/STING pathway also causes lethal autoimmune diseases such as Aicardi-Goutières Syndrome and STING-associated vasculopathy with onset in infancy (SAVI). Considering the important roles of TRIM38-mediated sumoylation in the regulation of the activation and stability of MDA5, RIG-I, cGAS and MITA/STING, this enzyme may serve as a potential target for drug development for autoimmune diseases.

CONCLUDING REMARKS

Recent studies have demonstrated multifaceted roles of TRIM38 in innate immune and inflammatory responses. TRIM38 acts as a SUMO ligase and targets RIG-I/MDA5 and cGAS/STING for SUMO1 modification, which is essential for their optimal activation and stability. Therefore, TRIM38 is essential for efficient innate immune responses to both RNA and DNA viruses. In these responses, TRIM38 functions with similar biochemical mechanisms, in that sumoylation of the RNA and DNA sensors by TRIM38 prevents their degradation. TRIM38 can also act as a ubiquitin ligase that targets TRIF for K48-linked polyubiquitination and degradation and therefore negatively regulates TLR3/4-mediated innate immune responses. In addition, TRIM38 can mediate lysosomal degradation of TAB2/3 in an enzymatic activity-independent manner to negatively regulate TNF/IL-1β-triggered inflammatory responses. Because TRIM38 is induced by type I IFNs and its inhibitory effects on TLR3/4-mediated or TNF/IL-1β-triggered inflammatory responses require its high expression level, it is possible that TRIM38 promotes innate immune responses at the early phase of viral infection, while inhibiting inflammatory responses at the late phase to avoid host damage. These multifaceted roles of TRIM38 qualify it as a critical regulator of proper innate immune and inflammatory responses against viral infection. One important question that remains unanswered is how TRIM38 is regulated to exert distinct enzymatic activities in different signaling pathways. In addition, the detailed mechanism on TRIM38-mediated lysosomal degradation of TAB2 and how TRIM38 is involved in autoimmunity are still elusive. Because innate immune response is essential for adaptive immunity, the function of TRIM38 in the activation of adaptive immunity should be of great interest. Further investigations into these and other outstanding questions will help to better explain the delicate regulatory mechanisms of innate immune and inflammatory responses and to evaluate whether TRIM38 is a proper target for drug development against infectious and autoimmune diseases.