Toll Like Receptor (TLR) Dysregulation

Background

Toll-like receptors (TLRs) are a family of 13 conserved transmembrane receptors that are at the forefront of directing innate and adaptive immune responses against invading bacteria, fungi, viruses and parasites (Akira 2003, Takeda, Akira 2004, Pasare, Medzhitov 2005, Tal, Adini et al. 2020, van der Made, Simons et al. 2020). Upon activation TLRs initiate overlapping and distinct signaling pathways in various cell types such as macrophages (MP), conventinal DC (cDC), plasmacytoid DC (pDC), lamina propria DC (LPDC), and inflammatory monocytes (iMO). Engagement of TLR with specific stressors (e.g. PAMPs and DAMPs) induces conformational changes of TLRs that allow homo- or heterophilic interactions of TLRs and recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM to control intracellular signalling pathways leading to the synthesis and secretion of appropriate cytokines and chemokines by cells of the immune system. TLRs have various biological roles both in pathogen combat and tissue homeostasis.

This KE is first developed in context of COVID-19 CIAO project.

The key gatekeepers in detecting and combating viral infections are TLR3, TLR7, TLR8 and TLR9 and these are predominantly localized in intracellular compartments. In the setting of COVID-19, multiple TLRs are likely relevant in viral combat. Literature covering TLR triggering via SARS-CoV-2 derived PAMPS (Pathogen Associated Molecular Patterns) include:

• TLR7 and TLR8 (+TLR3, TLR4, TLR6)  (Khanmohammadi and Rezaei, 2021)
• TLR1, TLR4 and TLR6 activated by SARS-CoV-2 spike proteins (Choudhury A et al, 2020)
• TLR9: Less CpG suppression in coronavirus compared to other viruses, for SARS-CoV-2 in the Envelope (E) open reading frame (E-ORF) and ORF10 (Ng et al., 2004; Digard et al. 2020) and multidisciplinary links described in Bezemer and Garssen, 2021

TLR dysregulation can be multi-fold:

1. Underperformance of TLR function leading to poor pathogen combat. This is covered in AOP 378

• COVID-19 patients having poor TLR function (due to polymorphisms) could potentially have less viral clearance capability and more adverse events leading to more severe disease and mortality. This has been shown for TLR7 loss of function polymorphisms (van der Made et al 2020). Knowledge Gap: it is not known if loss of function of other TLRs has a worse outcome in COVID-19 patients.

2. Overperformance of TLR function contributing to exaggerated immune response/cytokine storm/thrombosis/progression into ARDS and MOD. This is covered in AOP377

• TLR7 and TLR9 expression, measured by RNAseq gene analysis, is more elevated in black Americans than white Americans, which is proposed to explain in part the racial disparity in Covid-19 mortality rates via TLR mediated DC activation (Tal et al. 2020)
• genetic mutations leading to TLR9 gain of function in human is associated with immune-mediated disease and with a higher incidence of ICU acquired infection (Chatzietal.,2018;Ng et al.,2010).
• Higher presence of host derived TLR stressors in vulnerable patients can contribute to TLR overstimulation/dysregulation. (Bezemer and Garssen, 2021)

Different classes of "stressors" act on TLR activation/dysregulation

1.  Pathogen associated molecular patterns (PAMPs). TLRs can sense PAMPS during infection or upon exposure to stressors containing micro-organisms or fragments thereof (e.g. cigarette smoke, bioaerosols, house dust mite)

• TLR1 is activated by bacterial Lipopeptides
• TLR2 is activated by bacterial lipoproteins and glycolipids, TLR2 can form conformations with TLR1 and TLR6 to distinguish between diacyl and triacyl lipopeptides.
• TLR3 is activated by viral double stranded RNA(dsRNA)
• TLR4 is activated by Bacterial LPS
• TLR5 is activated by Bacterial flagellig
• TLR6 is activated by Bacterial lipopeptides and Fungal zymosan
• TLR7 and 8 recognize viral single stranded RNA(ssRNA) and bacterial RNA.
• TLR9 recognizes RNA and DNAmotifs that are rich in unmethylated Cytosine-phosphate-Guanine (CpG) sequences. CpG-motifs are higher expressed in the bacterial and viral genome compared to the vertebrate genome (Hemmi et al., 2000).

2. host derived Damage-Associated Molecular Patterns (DAMPS). Note that in the context and nomenclature of AOP these DAMPS cannot be labeld as "stressors" since they are derived from inside and not from outside, however these "pseudostressors" do act on the TLR receptors in similar way as PAMPs

• TLR2 and TLR4 are activated by heat shock proteins 60 and 70  (HSP60 and HSP70); extracellular matrix components (ECM); oligosaccharides of hyaluronic acid (HA) and heparan sulfate (HS) (Piccinini AM and Midwood KS, 2010).

• high-mobility group protein B1 (HMGB1) triggers TLR2, TLR4 and TLR9

• Oxidative injury/Oxidized phospholipids  triggers TLR4 mediated NET formation
• Human mitochondrial DNA (mtDNA), evolutionary derived from endosymbiont bacteria, contains unmethylated CpG-motifs and triggers inflammatory responses directly via TLR9 during injury and/or infection (Zhang et al., 2010).
• Altered self-ligands, called carboxy-alkyl-pyrroleprotein adducts (CAPs), that are generated during oxidative stress, are known to aggravate TLR9/MyD88 pathway activation (Zhanget al., 2010;Panigrahi et al., 2013). CAPs have been shown to promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo (Panigrahi et al., 2013).

3. synthetic TLR triggers/blockers (agonists/antagonists) for therapeutic purposes. Examples include CpG-ODNs triggering TLR9 for vaccin adjuvants/cancer treatment/immuno-modulation

Several Modulating factors can contribute to TLR activation/dysregulation

• Co-infection and Trauma (for instance ventilator induced damage) can induce increased levels of TLR9 stressor, mtDNA, which is known to drive worse outcome at ICU in setting of other disorders.  
• High levels of Visceral Fat, can increase TLR9 expression levels ánd circulating mtDNA
• Aging triggers both immunosenescence and inflammaging in part via impaired TLR function versus inappropriate triggering via increases of circulating DAMPS (Shaw et al 2011)
• Genetic polymorphisms can lead to TLR dysregulation (TLR9 gain of function and TLR7 loss of function with worse outcome at ICU Chatzi et al 2018, van der Made et al 2020, Chen et al 2011, )
• Circulating DAMPS such as mtDNA levels increase with age which is a familiar trait contributing to chronic inflammation, so called“inflamm-aging”in elderly people (Pinti et al., 2014).
• Vitamin D inhibits expression levels of TLR9
• Men, higher testosterone, higher TLR4

Patient specific Ex vivo analysis

• Levels of TLR specific stressors (for instance for TLR9, cell free DNA/RNA, mtDNA) are measurable in biological samples (serum, plasma)
• TLR gain of function and loss of function polymorphisms are measurable
• TLR expression levels on different cell types and different tissues are measurable by mRNA analysis and by protein analysis
• TLR function in response to stressors is measurable by analysing components of downstream cascades and read outs of inflammatory mediators (IL6, IL8, IL10, Il17, INF, TNFalpha, etc). This can be done by ex vivo stimulations of cells isolated from patients for instance PBMCs.

In vitro/ in vivo models

• TLR Reporter assays
• TLR knock-out mice

Cell applicability: TLRs are broadly expressed on various cell types. Examples include: epithelial cells, macrophages, neutrophils, platelets, dendritic cells, NK cells, Tcells, Bcells, neurons, Adipocytes.

Tissue/organ level : TLRs are broadly expressed in all vital tissues/organs: lung, heart, liver, spleen, kidney, brain, muscle, gut, skin

Taxonomic Applicability: TLRs are well conserved across species but between species variations are reported in terms of sensitivity towards stressors.  For instance certain CpG-ODNs have a stronger TLR9 activating potential in mice than in human and vice versa.

Life Stages: TLRs are expressed in all life stages but age variation of level of TLR activation/dysregulation are reported. In elderly immunoscenescence and inflammation are both linked to TLR dysregulation

Sex Applicability: Male and female subjects both express functionally active TLRs but sex differences have been reported. For instance certain TLR gain and/or loss of function polymorphisms have higher prevalence in men. Example of TLR7 loss of function (van der Made et al 2020) and TLR9 gain of function (Gao et al 2018, Traub et al 2012, Elsherif et al 2019). Higher testosterone in men has also been linked to higher TLR4 expression.

TLR7 is located in a region on the X-chromosome which have a high chance of escaping inactivation leading to higher expression levels in women. Estrogens trigger TLR7, which is higher in women. Exposure of Peripheral blood mononuclear cells (PBMC) to TLR7 ligands will cause a higher production of type I IFN (IFN-a) in female cells than male cells.  (Kovats, 2015;  Takahashi and Iwasaki, 2021; Libert et al.,  2010; Scully et al., 2020)

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