Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Å limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex.The carboxy-terminal domain of the BBK32 protein from  Borrelia burgdorferi sensu lato complex, is the most common tick-borne infection in the Northern hemisphere. As a pathogen that is transmitted within blood and body fluids, Borrelia species must combat the complement system, which is a soluble host defense pathway that marks invaders for elimination. Previously, we demonstrated that the B. burgdorferi protein BBK32 blocks the activation of the classical pathway of complement by binding to and inhibiting the initiating protease, C1r. Here we show that the BBK32 orthologue from B. garinii, designated BGD19, is unable to neutralize complement-mediated serum killing. In contrast, BBK32 and the B. afzelii orthologue BAD16 potently inhibits serum killing. Comparison of the amino acid sequences of these proteins identified three residues unique to BGD19 that contribute to its reduced inhibitory activity. The three-dimensional structure of BBK32-C, reported here, showed that these residue positions are surface exposed in BBK32, further supporting their role in C1r inhibition. These results render important insight into how Lyme disease-associated spirochetes recognize and block an important human innate immune pathway and provide mechanistic insight into evolutionarily optimized C1r inhibitors.Lyme disease, caused by  Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis and include B. burgdorferi sensu stricto, B. garinii, and B. afzelii. B. burgdorferi sensu stricto (referred to as B. burgdorferi hereafter) causes 300,000 cases of Lyme disease in the United States each year [B. garinii and B. afzelii are the most common etiological agent of Lyme disease in Europe and Asia [B. burgdorferi sensu lato are the leading arthropod-borne infectious agents in the Northern hemisphere and are capable of hematogenous dissemination whereby a wide range of remote host tissues are colonized. To survive and persist in immunocompetent hosts, Lyme-associated spirochetes must evade host immune defenses including the evolutionarily ancient proteolytic cascade of innate immunity known as the complement system.Spirochetes belonging to the ach year , while Band Asia . B. burgComplement is a group of nearly three dozen proteins that combine to coordinate a tightly controlled set of proteolytic reactions directed at target cell surfaces ,4. ComplB. burgdorferi sensu lato species themselves are known to encode up to five distinct proteins  that bind to and recruit factor H to the bacterial surface, thereby hijacking its complement protective activities [B. burgdorferi sensu lato proteins have now been identified that exhibit specific complement inhibitory activities [Host cells express several proteins which function to regulate complement and thus are typically protected from unintended targeting by the cascade. In contrast, pathogens that traffic in fluids where complement is present at high concentrations have necessarily evolved mechanisms to evade complement detection and activation. For example, many human pathogens secrete membrane-associated proteins which bind endogenous host regulators of complement . In thistivities –13. In ativities –16, recrtivities –20, or btivities ,22. In ttivities .B. burgdorferi BBK32 remains the lone identified and characterized classical pathway-specific inhibitor [S1 Fig). C1 thereby functions as both the pattern recognition molecule and initiating zymogen of the classical pathway. The C1 complex circulates in blood in an inactive form until C1q is recruited to the surface via recognition of receptors such as immune complexes. C1q-binding promotes autocatalytic activation of the C1r protease within the C1 complex which then cleaves C1s to form fully activated C1. At this step the C1s enzyme cleaves complement components C2 and C4, and the classical pathway intersects the lectin pathway at the formation of classical/lectin pathway C3 convertases (C4b2a). C3 convertases then convert complement C3 into its activated forms, which in turn drive downstream reactions of the cascade. Previously we have shown that the C-terminal globular domain of B. burgdorferi BBK32 (termed BBK32-C) blocks classical pathway activation by binding with high-affinity to the initiating serine protease C1r, and preventing both its autocatalytic and C1s cleavage activities within the C1 complex [.Within the small arsenal of borrelial complement inhibitors, the surface-expressed lipoprotein nhibitor . The cla complex  B. burgdorferi sensu lato species B. garinii and B. afzelii to better understand the structural and mechanistic basis for BBK32-mediated C1r inhibition. Herein we report the first crystal structure of the anti-complement domain of BBK32 which reveals a novel helical bundle fold. A chimeric mutagenesis strategy provided additional insight into the reduced in vitro activities of B. garinii BBK32 orthologue BGD19 relative to the B. afzelii BBK32 orthologue BAD16 and B. burgdorferi BBK32 itself. Biochemical studies were then used to map the BBK32 binding site on human C1r and demonstrated that the serine protease (SP) domain was required for BBK32/C1r complex formation. The results of this study significantly improve our understanding of the unique classical pathway inhibition properties of BBK32 and suggest that BBK32-mediated complement evasion activity is shared across major species of Lyme disease-associated spirochetes.In this study we investigated the activity of BBK32 orthologues encoded by the prevalent B. burgdorferi BBK32 is a surface-exposed lipoprotein with multiple roles in vertebrate host interactions [B. burgdorferi adhesin and has been shown to play critical roles in colonization and dissemination [Fig 1A). Orthologues to B. burgdorferi BBK32 within the sensu lato complex are encoded by B. afzelii (termed BAD16) and B. garinii (termed BGD19). These BBK32 orthologues share ~90% and ~70% overall amino acid sequence identity relative to B. burgdorferi BBK32, respectively .ractions ,24–32. Nractions  and lateractions . Owing tmination ,33. Whilmination ,31,34,35mination  B. burgdorferi sensu lato complex in vitro studies have shown that B. garinii is more sensitive to the lytic component of human serum (i.e. the complement membrane attack complex) than B. burgdorferi or B. afzelii [B. garinii does not recruit functional human factor H via factor H-binding proteins, unlike human serum-resistant B. burgdorferi and B. afzelii [B. garinii strains are also human serum sensitive in a C1q-dependent manner, indicating a potential role for the classical complement pathway in the human serum susceptibility phenotype of B. garinii [B. garinii BGD19 and B. afzelii BAD16 are unknown. To address this, we produced recombinant proteins of each orthologue corresponding to the C-terminal complement inhibitory region of BBK32 (i.e. BBK32-C) , and evaluated their ability to bind to human C1 complex and isolated human C1r using surface plasmon resonance (SPR). These experiments show that BAD16-C and BGD19-C each bind to human C1 and C1r with high affinity (S1 Table).Among the  afzelii . Increas afzelii . However garinii . In this50, C3b deposition = 32 nM; IC50, MAC deposition = 35 nM) exhibited a two to three fold decrease in potency relative to BBK32-C , whereas BAD16-C exhibited no greater than a two-fold increase in potency   based on non-overlapping 95% confidence intervals (S2 Table). In a classical pathway hemolysis assay we found a larger difference in relative potency as concentrations of B. garinii BGD19 up to 1 μM failed to exhibit saturable inhibition of classical pathway activation in hemolysis assays , unlike BBK32 (IC50 = 170 nM) or BAD16 IC50 = 55 nM) which both exhibit dose-dependent protection of sheep red blood cell lysis by human serum (S2 Table).Previously we have shown that the high-affinity interaction formed between BBK32-C and human C1r correlates with blockade of classical pathway activation in assays where human serum is used as the source of complement . In a clFig 1B). In B. burgdorferi BBK32 and B. afzelii BAD16 these residues are Glu-308, Gln-319, and Glu-324, whereas in B. garinii BGD19 these positions are changed to Lys-308, Lys-319, and Gln-324. To investigate the potential role of these residues in mediating C1r inhibition, we produced a chimeric BBK32-C protein, termed BXK32-C, where each residue was changed to the B. garinii BGD19 residue (i.e. BBK32-E308K-Q319K-E324Q). Interestingly, the inhibitory activity of the chimeric BXK32-C shifts from that of BBK32 to BGD19 . These data indicate that residues encoded at one or more of these positions in BGD19 likely contribute to its observed reduction in human classical complement pathway inhibitory activity.Sequence alignment of BBK32, BAD16, and BGD19 reveals there are just three non-conservatively substituted amino acids shared between BBK32-C and BAD16-C that are different in BGD19-C  [B. garinii bgd19), and B314/pBAD16 (B. afzelii bad16) . BBK32, BGD19 and BAD16 were expressed heterologously in B. burgdorferi strain B314 and surface localization was assessed using the proteinase K accessibility assay. Here, BAD16 and BGD19 were sensitive to protease digestion under conditions that the subsurface endoflagellar protein, FlaB, was not affected, indicating that the BBK32 orthologues were surface exposed and that the borrelial cells were structurally intact, respectively . Additionally, we assessed the different transcript levels of the bbk32 orthologues by qRT-PCR analysis and found that all orthologues were expressed at levels that were not significantly different . Next, Far Western blot experiments were performed using biotinylated-human C1  or human C1r  as probes and normalized to the levels of B. burgdorferi BBK32 produced. These data suggest that BGD19 and BAD16 bind with similar affinity to human C1 . However, when C1r is used as the probe, BGD19 binds only weakly and not significantly different relative to a vector only control (denoted as “Vector”), whereas BBK32 and BAD16 each bind to C1r .Next, we investigated the activity of BGD19 and BAD16 when expressed as full-length lipoproteins on the spirochetal surface by using the poorly adherent, non-infectious strain B314 of i bbk32) , B314/pBFig 4) BGD19 showed significantly reduced C1r binding relative to BBK32 and BAD16 . Next, we assessed the ability of heterologously expressed BGD19 and BAD16 to confer serum resistance to strain B314. While expression of BBK32 and BAD16 at the B314 surface protected spirochetes from classical pathway-mediated complement killing, BGD19 did not significantly reduce killing relative to the vector only control . Interestingly, BAD16 exhibited greater resistance to serum relative to BBK32 .To assess the binding of C1r to native, surface exposed BAD16 and BGD19 relative to BBK32, we incubated whole, intact borrelial strain B314 cells with immobilized C1r. In agreement with the whole cell lysate assays ((206–348)) which produced protein crystals . Importantly, the BBK32(206–348) construct retained full C1-binding, C1r-binding and complement inhibitory activities .Circular dichroism studies indicate that the secondary structure of BBK32-C is predominantly helical in solution . Beyond (206–348) crystals grew in space group P65 with one molecule per asymmetric unit and diffracted to 1.7 Å resolution (Table 1). BBK32(206–348) consists of five α-helices and adopts a helical bundle fold . Starting from the N-terminus, helix α1 (residues Ser-211 to Met-245) interacts with helices α3 (Lys- 256 to Ala-286), α4 (Ile-293 to Lys-317), and α5 (Leu-323 to Ile-347) to form an anti-parallel four-helix bundle. Helix α2 (Asn-251 to Ala-261) does not participate in the core bundle motif but rather forms hydrophobic interactions with helix α1 and sits at an angle of ~120° relative to helix 3. Adaptive Poisson-Boltzmann Solver software was used to calculate the electrostatic potential of the BBK32-C molecular surface [Fig 6B). The protein surface is characterized by several contiguous positively charged regions with a larger negatively charged surface being formed where the C-terminal and N-terminal helices meet. Overall the structure of BBK32-C is best characterized by a positively charged anti-parallel four helix bundle where a fifth helix, helix α2, protrudes away from the helical core.BBK32 surface  Fig 3). In the BBK32-C structure E308 is a surface exposed residue located midway through α4, while Q319 and E324 are in the short loop connecting α4 and α5. Q319 and E324 are also surface exposed and together present a contiguous surface region . Homology models of BAD16-C, BGD19-C, and BXK32-C were constructed using SWISS-MODEL and templated on the BBK32-C crystal structure . These models predict that residues at the 308, 319, and 324 positions (BBK32 numbering) [S5 Fig). As each of these residues is exposed to solvent, they are potentially positioned to interact with C1r, supporting the functional data implicating their involvement in BBK32-mediated complement inhibitory activity .The relative activities of the BXK32-C mutant suggest that one or more of the non-conservative amino acid substitutions between BBK32/BAD16 and BGD19  contribute to C1r inhibitory activity (mbering) ,41 are a(206–348) and identification of surface residues which affect C1r inhibition presented above provides insight into the structural determinants for BBK32-mediated C1r recognition. However, the C1r domains involved in mediating BBK32/C1r complex formation are unknown. C1r is a 92 kDa chymotrypsin-like serine protease with a modular architecture consisting of two complement C1r/C1s, Uegf, Bmp1(CUB) domains, an epidermal growth factor (EGF) domain, two complement control protein (CCP) domains, and a serine protease (SP) domain arranged in sequential fashion (CUB1-EGF-CUB2-CCP1-CCP2-SP) . We noted that purified full-length C1r has been reported to undergo a series of autoproteolytic cleavages when incubated for prolonged periods [Fig 8A, black line). The chromatogram displayed three well-resolved peaks . Next, purified BBK32(206–348) was injected alone resulting in a single peak (labeled 4) . Finally, a 2-fold molar excess of BBK32(206–348) was mixed with the autoproteolytic C1r digestion reaction and injected onto the column ( blue dashed line). While peaks 1, 2, 4, and 5 remain, a new peak also appears (labeled peak 3). Analysis of each peak in the BBK32(206–348)/C1r digestion injection was performed using non-reducing SDS-PAGE . A single band which migrates on the gel at an apparent molecular mass of 55 kDa was found in peak 2. This same 55 kDa band co-eluted with BBK32(206–348) in peak 3. Mass spectrometry analysis identified the 55 kDa band observed in peaks 2 and 3 to be identical to one another and to correspond to residues Leu-300 to Asp-705 of C1r. This region maps to the C-terminal portion of C1r and includes the C-terminal six residues of the CUB2 domain and the entirety of the CCP1-CCP2-SP domains (hereafter referred to as C1rCCP1-CCP2-SP-auto). The C1rCCP1-CCP2-SP-auto fragment identified here closely matches the previously reported autolytic C1r fragment known as γ-B [(206–348) by mass spectrometry analysis.The crystal structure of BBK32 periods ,43. Folln as γ-B ,43. As e(206–348) with the C1rCCP1-CCP2-SP-auto proteolytic fragment suggested that BBK32 binds to the C-terminal region of C1r. To confirm this, we purified C1rCCP1-CCP2-SP-auto and used SPR to assay its affinity for BBK32. Indeed, this autolytic C1r fragment displays similar affinity for BBK32 (KD = 1.5 nM) to that previously measured for full-length C1r . To further refine the mapping of the BBK32 binding site on C1r we produced recombinant C1r domain truncations corresponding to the C-terminus of C1r. While C1r-CCP1 and C1r-CCP1-CCP2 failed to interact with BBK32 in SPR binding experiments, a construct containing only the CCP2-SP domains bound with similar affinity to BBK32  as was found for C1, C1r, and C1r CCP1-CCP2-SP-auto. Efforts to produce a recombinant protein corresponding to the C1r SP domain only were unsuccessful. However, collectively the data presented above strongly suggests that the SP domain of C1r is required for high affinity interaction with BBK32. A model of full-length C1r was constructed from the available C1r domain truncation mutants crystal structures [Fig 8E).Co-migration of BBK32ructures –46, and Borrelia burgdorferi sensu lato complex are among a group of human pathogens that have evolved several mechanistically distinct extracellular complement inhibitor proteins [B. burgdorferi sensu lato species employ a well-known pathogenic anti-complement strategy via expression of proteins which recruit the endogenous host regulator of the alternative pathway of complement, factor H, to the bacterial surface [Borrelia also produce a distinct set of surface proteins capable of binding host plasminogen and specifically degrading complement components [Human complement is an evolutionarily ancient arm of the innate immune system that was first described nearly 120 years ago. Historically, complement has been regarded as a ‘first-line-of-defense’ against invading pathogens. Indeed, if a pathogen is unable to evade detection by one of the three complement pathways, initiating serine proteases begin converting zymogen complement proteins into activated fragments resulting in distinct but synergistic host defense mechanisms that include: i) opsonization ; ii) phagocyte recruitment C3a, C5a); iii) priming of the adaptive immune system ; and iv) lysis (membrane attack complex). In this context it is no surprise that microorganisms that encounter blood, and other complement containing fluids, have evolved mechanisms to evade complement recognition and activation. Lyme disease spirochetes of the a; iii) pproteins ,22. For  surface ,48. Lymemponents –20, protmponents ,49, and mponents –16.B. burgdorferi infection compared to wild type mice [B. burgdorferi infections. Future studies will be important to elucidate the in vivo role of BBK32-mediated classical pathway complement inhibition on the subversion of T-dependent B cell responses by Lyme disease-causing spirochetes.While complement has traditionally been viewed as a sentinel against microbial intruders, it is no longer considered an isolated innate immune response. Complement is integral to homeostatic maintenance and has direct roles in the regulation of both T cell and B cell immunity –52. Inteype mice . This isype mice ,56. GiveB. burgdorferi and B. afzelii have been classified as resistant whereas B. garinii strains have often been classified as sensitive [Borrelia burgdorferi sensu lato species to complement may reflect an in vivo selection process that contributes to the pathogens ability to colonize different reservoirs [Peromyscus leucopus, is the natural reservoir for B. burgdorferi in the Midwest and northeastern United States [B. afzelii and B. garinii, respectively [B. burgdorferi (strain B31) and B. afzelii (strain PGau), as well as a human serum-sensitive strain of B. garinii (strain IP90), to investigate the relative complement inhibitory activities of BBK32 orthologues. Quantitative affinity measurements using SPR with purified proteins indicated that recombinant BGD19-C binds C1r with similar affinity to that of BBK32-C and BAD16-C . Surprisingly, BGD19-C showed slightly weaker inhibition in complement assays involving artificial surfaces , and conferred significantly reduced protection from complement-mediated lysis to the naïve membranes of sheep red blood cells . These results suggest that binding of recombinant C-terminal BBK32 orthologues is necessary but not sufficient for potent C1r inhibitory activity. However, we note that when full-length BGD19 was expressed on the surface of a surrogate B. burgdorferi strain, it bound C1r weakly in qualitative binding assays relative to either full-length BBK32 or BAD16. It is unclear why C1r-binding observed in these assays differs from the similar affinities measured with recombinant proteins using SPR. Nonetheless, consistent with the weaker complement inhibitory properties of recombinant BGD19-C, surface expressed full-length BGD19 failed to protect B. burgdorferi B314 from complement-mediated killing . Collectively, our results show that B. garinii BGD19 has significantly reduced capacity to inhibit in vitro classical pathway complement activation compared to B. burgdorferi BBK32 or B. afzelii BAD16.In regards to human serum susceptibility, ensitive . Differeservoirs –59. For d States  whereas ectively . For thiB. garinii to human serum killing is related to the reduced activity of borrelial complement evasion proteins, as has been previously proposed for B. garinii factor H-binding proteins [in vitro serum-sensitivity classification scheme is recognized as being dependent on reagents, experimental conditions, and importantly the strains being studied [B. burgdorferi and B. afzelii, B. garinii also causes human infections, and thus, some B. garinii strains can overcome complement-mediated clearance in vivo. Borrelia burgdorferi sensu lato spirochetes, including B. garinii, likely have multiple layers of functional redundancy that make up its complement evasion repertoire in vivo. Thus, while the reduction in activity of a single complement inhibitor like the BBK32 orthologue BGD19 is expected to contribute to the relative ability of B. garinii to survive complement-mediated attack in vivo, it must be considered in the context of a functionally redundant borrelial complement evasion system. Ultimately it will be the collective activities of these inhibitors, rather than dominance by a single complement evasion molecule, that would be expected to drive in vivo susceptibility of Borrelia burgdorferi sensu lato spirochetes to complement.Our data support the notion that the relative increased susceptibility of proteins . However studied . Like B.B. burgdorferi BBK32-C presented here has provided the first insight into the structural determinants required for high affinity C1r interaction and inhibition by borrelial BBK32-like proteins. We probed the BBK32 molecular surface using a chimeric BBK32-C construct encoding three non-conserved surface residues originating from BGD19 (i.e. BXK32-C) and found that these substitutions alone shift the inhibitory activity of BBK32 towards that of BGD19. BBK32-like sequences are unique to the Borrelia genus and include three families of proteins found in relapsing fever-associated spirochetes termed FbpA, FbpB, and FbpC [B. burgdorferi sensu lato orthologues and ranges between 25% and 60% identity at the amino acid level. Our data indicate that subtle changes in amino acid sequences can result in significant differences in the ability of B. burgdorferi sensu lato BBK32 orthologues to block human complement and thus it will be important to determine if BBK32-like classical pathway complement inhibition is restricted to Lyme disease spirochetes or is common to all pathogenic Borrelia.The crystal structure of and FbpC . The relB. burgdorferi BBK32, as well as its orthologues, have unique and apparently disparate functions within the vertebrate host. The disordered N-terminal half acts as an adhesin by binding to glycosoaminoglycans and the extracellular matrix protein fibronectin, while the ordered carboxy terminal half acts as a C1r-binding complement inhibitor . While discrete BBK32-binding sites have been identified for each of these host ligands, it remains unknown if BBK32 interacts simultaneously with C1r and fibronectin or GAGs. This may be of importance as the formation of functionally synergistic ternary complexes involving virulence factors from other pathogens, such as Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) in complex with complement C3 and fibrinogen, have been described [Borrelia–or even in other human pathogens–has yet to be fully evaluated.escribed . Furtherescribed –65. WhetAmong the multi-pronged borrelial complement evasion arsenal , BBK32 iB. burgdorferi B31 strains ML23 and B314, as well as B. afzelii strain PGau and B. garinii strain IP90, were grown in BSK-II media supplemented with 6% normal rabbit serum  under microaerobic conditions at 32˚C, 1% CO2 atmosphere, pH 7.6. Strain B314 is a serum-sensitive, non-infectious strain B31 derivative that lacks most linear plasmids [B. burgdorferi cells were enumerated by dark field microscopy.plasmids ,68. All bbk32 genes from B. afzelii strain PGau and B. garinii strain IP90, designated as bad16 and bgd19, respectively, were cloned into the shuttle vector pBBE22luc. To carry this out, oligonucleotide primers were designed based on the sixteenth open reading frame of lp17 from B. afzelii strain PKo  and the nineteenth open reading frame of lp17 from the B. garinii strain PBr . The letter “D” or “d” used to denote the orthologous protein or gene, respectively, is due to their presence on the lp17 episome, which is referred to as the “D” plasmid in B. burgdorferi strain B31 [B. afzelii strains are 100% identical whereas the B. garinii proteins share 96% identity. Oligonucleotide primers were synthesized by Eurofins, Inc.  and their corresponding sequences are shown in Table 2. Oligonucleotide primers with sequences that overlapped with the borrelial gene and the vector pBBE22luc were used for PCR amplification using genomic DNA from B. afzelii strain PGau and B. garinii strain IP90 as template. The amplified fragments contained 395 and 491 bp of upstream sequences and 185 and 177 bp downstream from the translational start site and stop codon corresponding to the 1059 bp bad16 and 1065 bp bgd19 genes, respectively. The resulting PCR products were 1639 bp and 1733 bp for bad16 and bgd19, respectively. The plasmid pBBE22luc was digested with BamHI HF and SalI HF  and assembled separately with each of the aforementioned PCR fragments using the manufacturer’s instructions for NEBuilder (New England Biolabs). The resulting constructs were transformed into Escherichia coli DH5α cells (F–ϕ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 phoA supE44 λ–thi-1 gyrA96 relA1) and transformants selected on LB agar plates containing kanamycin at 50 μg/ml . The resulting constructs, which contained bad16 and bgd19 expressed under the control of their native promoters, were confirmed by sequencing and designated pBAD16 and pBGD19, respectively.Heterologous rain B31 . Note thluc vector. The cloning was confirmed with PCR and sequencing with primers pncAf and lucf (.Transformation of strain B314 with pBAD16 and pBGD19 was done as previously described . TransfoB. burgdorferi strain B31), BBK32-C(206–348) , BGD19-C , BAD16-C , and BXK32-C (i.e. BBK32-C E308K-Q319K-E324Q) were sub-cloned into the pT7HMT vector [(206–348), a DNA fragment encoding BBK32 residues 206 to 348 was generated by PCR from the existing pT7HMT-BBK32-C plasmid [BamHI and NotI sites at the 5’ and 3’ends, respectively. For BGD19-C, BAD16-C, and BXK32-C an Escherichia coli codon-optimized DNA sequence flanked with a 5’ BamHI site, a 3’ NotI site, and a stop codon were synthesized commercially using IDT Technologies gBlock Gene Fragment service. Recombinant C1r domain truncations corresponding to the CCP1, CCP1-CCP2, or CCP2-SP domains (as defined by UNIPROT ID: P00736) were produced as follows. Synthetic E. coli codon-optimized DNA (synthesized by IDT Technologies gBlock), corresponding to the sequence of human C1r domains CCP1-CCP2-SP (amino acid residues 307–705), was used as a PCR template along with oligonucleotide primers that appended BamHI and NotI at the 5’ and 3’ends, respectively, to produce DNA fragments corresponding to CCP1 (residues 309–373), CCP1-CCP2 (residues 309–449), and CCP2-SP (residues 376–705). All C1r domain truncations were purified under denaturing conditions using previously published protocols [Recombinant BBK32-C , 140 mM NaCl, 0.005% Tween-20). Proteins were immobilized using standard amine coupling chemistry on CMD200M biosensor chips (Xantec) as described previously [CCP1-CCP2-SP-auto analyte over BBK32(206–348) . The C1 and C1r injection series were performed in HBS-T buffer supplemented with 5 mM CaCl2. C1 injections consisted of a twelve point, two-fold dilution series ranging from 0 to 150 nM C1 for 2 min association and 3 min dissociation. C1r was injected using a single-cycle kinetic format [CCP1-CCP2-SP-auto, C1rCCP1, C1rCCP2, and C1rCCP1-CCP2-SP injections were identical to that of C1r using a concentration range of 0.8 to 500 nM. Kinetic analysis was performed for each set of sensorgrams injections using T200 Evaluation Software  using a 1:1 (Langmuir) binding model and a dissociation constant (KD) was calculated from the resulting fits. All injection series were performed in triplicate and the mean value is reported for each KD ± standard deviation.All SPR experiments were conducted on a Biacore T200 instrument at 25°C and unless otherwise noted using a flowrate of 30 μl mineviously . The folc format  using a The ability of recombinant BBK32 orthologue proteins to inhibit the activation of human complement was assessed using two assay formats. First, an ELISA-based assay was used that relies on the activation of the classical pathway via surface-immobilized IgM (Athens Research and Technology) and subsequent detection of the complement deposition products derived from normal human serum (Innovative Research), specifically C3b or MAC through use of monoclonal antibodies (both purchased from Santa Cruz Biotechnology) . Each boB. burgdorferi strains B314/pBAD16 and B314/pBGD19 were grown in complete BSK-II media, harvested by centrifugation at 5,800 x g, and washed twice with PBS. The cell pellet was re-suspended in 0.5 ml of either PBS alone or with PBS with proteinase K (Invitrogen) to a final concentration of 200 μg ml-1. All samples were incubated at 20°C for 40 min. Reactions were terminated by the addition of phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1 mM. Cells were again pelleted by centrifugation , washed twice with PBS containing 1 mM PMSF, re-suspended in Laemmli sample buffer, and resolved on SDS-PAGE. The separated proteins were transferred to a PVDF membrane (Thermo Fisher Scientific) and probed as described below with anti-BAD16, anti-P66, and anti-FlaB antibodies, respectively.B. burgdorferi adherence assay was done as previously described with slight modifications [B. burgdorferi strains B314/pBBE22luc (vector only control), B314/pCD100 (expresses bbk32), B314/pBAD16 (expresses bad16), and B314/pBGD19 (expresses bgd19) were grown to mid-logarithmic phase at 32°C, 1% CO2, pH 7.6. All B. burgdorferi strains were subsequently diluted to 107 organisms/ml in BSK-II medium without serum. The resulting B. burgdorferi samples, in 0.1 ml volumes, were applied onto the coverslips and incubated for 2 hr at 32°C. Unbound bacteria were removed from the coverslips by gentle washing with PBS; this wash step was repeated 7 times. The coverslips were applied to a glass slide and the binding of spirochetes was scored by dark field microscopy.ications ,75. BrieB. burgdorferi strains were grown to exponential phase at 32°C, 1% CO2, pH 7.6, and 80 μl of a 106 cell suspension in BSK-II medium was added to 20 μl of normal human serum  to give a final volume of 100 μl . The samples were placed in microtiter plates and the suspensions were sealed and incubated at 32°C for 2 h. Heat-inactivated normal human serum (hiNHS) was used as a control. After incubation, B. burgdorferi cells were scored by dark field microscopy and the percentage of viable B. burgdorferi cells were calculated from randomly chosen fields and based on immobilization, loss of cell envelope integrity, and/or overt lysis.Complement sensitivity assays were performed as previously described . Briefly(206–348) was concentrated to 5.1 mg ml-1 in a buffer of 10 mM HEPES (pH 7.3), 50 mM NaCl. Crystals of BBK32(206–348) were obtained by vapor diffusion of sitting drops at 20°C. Drops were setup by mixing 1 μl of protein solution with 1 μl of precipitant solution. Two crystallization conditions were identified. The first condition contained 0.1 M MES (pH 6.5), 0.2M ammonium sulfate, and 30% PEG-MME 5,000. Small plate clusters reproducibly appeared in this condition between 2 and 5 d with rounds of microseeding producing large plates which could be harvested and cryoprotected with supplementation of 5% glycerol to the precipitant solution. Crystals in this condition grew in the space group P21 with four BBK32(206–348) molecules in the asymmetric unit, diffracting to 2.5 Å resolution. A second condition was identified containing 15% PEG 3,350 and 0.1M succinic acid (pH 7.0). These crystals appeared only after prolonged incubation . Cryoprotection was achieved by supplementing the precipitant solution with 20% glycerol. These crystals grew in space group P65 with a single copy of BBK32(206–348) in the asymmetric unit, diffracting to 1.7Å. Monochromatic X-ray diffraction data were collected at 0.973-Å wavelength using beamline 22-ID of the Advanced Photon Source . Diffraction data were integrated, scaled, and reduced using the HKL2000 software suite [P21 dataset failed. However, a single solution was found using the P65 dataset by the MRage program [work/Rfree). Subsequent manual building was performed using COOT [work/Rfree). Residues 206–208 are missing from the final model due to poor electron density and thus the completed model contains BBK32 residues 209–348. Using the final refined model obtained from this crystal as a molecular replacement search model readily provided a solution for the P21 crystal and a final refined model of 20.8% / 26.9% (Rwork/Rfree). Refined coordinates and structure factors for the P65 crystal form have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University (www.rcsb.org/) under PDB ID code 6N1L. A description of crystal cell constants, diffraction data quality, and properties of the final model for BBK32(206–348) can be found in Table 1. Representations of protein structures and electron density maps were generated by PyMol (www.pymol.org/). Homology models of BGD19-C, BAD16-C, and BXK32-C presented in BBK32re suite . Of all  program  implemen program –80. MRag program . The top program . Despiteing COOT  and iter-1 was diluted into an equal volume of 50 mM Tris (pH 8.0), 0.5 mM CaCl2 and allowed to incubate overnight at 37°C. 150 μl of this reaction was mixed with either 100 μl of buffer or 100 μl of BBK32(206–348) at 2 fold molar excess relative to full-length C1r. A third sample was prepared with only BBK32(206–348). Each 250 μl sample was then injected onto a SuperDex 200 Increase 10/300 GL small scale size-exclusion column  previously equilibrated in 10 mM HEPES (pH 7.3), 140 mM NaCl at a flowrate of 0.5 ml min-1. Peaks were evaluated by SDS-PAGE under non-reducing conditions. The bands in ‘Peak 3’ (elution volume 14.5 to 15.5) were identified by mass spectrometry.A total of 400 μl of purified C1r (Complement Tech) at 1.0 mg ml(206–348) were excised from the SDS-PAGE gel, reduced and alkylated, and digested with trypsin overnight by standard methods. Extracted peptides from each digest were subjected to LC-tandem MS analysis for verification and determining the coverage of C1r in the 55kDa gel band. The digests were resolved by reversed phase nanoLC in a C18 column , in a 1% to 35% acetonitrile, 100 minute gradient , eluting into a Q Exactive Plus MS system. Data were acquired in data dependent mode, MS scans at 35k, with 16 dependent MS2 scans per cycle at 17.5k resolution. The HRMS data files were searched using Mascot version 2.6 against a custom database consisting of the full length native human C1r sequence (Uniprot accession P00736), and a sequence for BBK32(206–348) consistent with the predicted amino acid sequence of the cloned construct. Peptides considered were restricted to semi-Trypsin specificity, with tolerances of 10ppm (MS) and 0.01Da (MS2 fragment) allowed, with fixed Carbamidomethyl (Cys), and variable modifications Deamidation  and Oxidation (Met) included. Peptides identified at P > 0.05 threshold were manually inspected to verify the quality of the apparent sequence coverage.Gel bands representing the autolytically formed 55kDa form of C1r, and BBK32B. burgdorferi strains, B314 (no vector), B314/pBBE22luc (vector only control), B314/pCD100 (produces BBK32), B314/pBAD16 (produces BAD16), and B314/pBGD19 (produces BGD19). The protein lysate of one repeat of each B. burgdorferi strain was resolved in the same SDS–PAGE gel and transferred to PVDF membranes as described [In the Far Western assay, three biological repeats were performed for each escribed . PVDF meB. burgdorferi strain B31 FlaB (Affinity BioReagents) was diluted at 1:4,000 and incubated with the blot for 1 hr. After washing in PBS, 0.2% Tween-20, the blot was next incubated with a 1:10,000 dilution of Goat anti-mouse IgG with IRDye 680RD (Li-Cor Biosciences) as the secondary antibody. The membrane was washed extensively in PBS, 0.2% Tween-20 and scanned using the Li-Cor Odyssey Fc Imaging System.Immunoblotting to detect the endoflagellar antigen FlaB was done for all samples using the same PVDF membrane used in C1 or C1r Far Western detection. A monoclonal to The signals obtained from the Li-Cor unit were analyzed using the Image Studio-lite Ver 5.2.5 software. The bands were detected with manual adjustment to their shape relative to background. All BBK32 orthologue signals obtained were initially normalized to the FlaB signal from the same sample, then to the B314/pCD100 on the same blot, to compare across distinct Far Western blots quantitatively as indicated here for BAD16:The resulting values were then used in statistical analyses described below.Conventional immunoblots were also performed with rat polyclonal antibodies to BBK32-C, BAD16-C, and BGD19-C  produced in the B314 background strain. To detect membrane-bound immune complexes, Goat anti-Rat IgG with an IRDye 800CW conjugate (Li-Cor Biosciences) was used as a secondary antibody and diluted to 1:10,000. Detection of P66 was accomplished using rabbit anti-P66 serum (generously provided by Sven Bergström) diluted 1:1000 followed by detection on membrane immune complexes using a 1:10,000 dilution of Goat anti-rabbit conjugated with IRDye 800CW (Li-Cor Biosciences). The membranes were scanned using the Li-Cor Odyssey Fc Imaging System.B. burgdorferi strain B314/pCD100 (expresses bbk32), B314/BGD19 (expresses bgd19), and B314/BAD16 (expresses bad16) were grown to the exponential growth phase , and total RNA was isolated from 5 x 108 cells using Direct-zol RNA MiniPrep . The RNA samples were treated with the in-kit DNase I and TURBO DNA free kit  to eliminate contaminating DNA. RNA integrity was examined by gel electrophoresis.Three independent cultures of each flaB and bbk32 via quantitative RT-PCR (qRT-PCR) were adopted from prior studies [bad16 and bgd19 were designed in this study and shown in B. burgdorferi sensu lato strains as template. Reverse transcription reactions of three biological repeats of each strain were carried out with SuperScript II Reverse Transcriptase . A control reaction with a mixture lacking reverse transcriptase was performed for each primer set to confirm that DNA was not present. Subsequently, the products from the reverse transcription reaction were subjected to quantitative real-time PCR using an Applied Biosystems StepOnePlus Real-Time PCR system. PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) was used to perform quantitative PCR in triplicate . The constitutively expressed flaB gene of B. burgdorferi was used for normalization as previously described [bbk32 orthologues were first normalized to the flaB in the same sample, then the normalized values of bad16 and bgd19 were compared to the level of bbk32 using the 2-ΔΔCt method. The final fold differences were used in statistical analyses.Oligonucleotide primers for amplifying  studies  and primescribed ,86. The 50 values in ELISA and hemolytic complement assays using recombinant proteins, non-linear regression was performed using a variable four-parameter fit where the top and bottom values were constrained to 100 and 0, respectively. Two-way ANOVA were used in C1r binding assay and serum sensitivity assay, and One-way ANOVA were used in Far Western and qRT-PCR analyses.Statistical analysis was performed with GraphPad Prism version 7. For calculation of ICS1 FigBorrelia species, BBK32, or active orthologues of BBK32, can block activation of C1r and inhibit the classical complement cascade. B) A model for BBK32-mediated inhibition of the classical pathway. C1 complex, consists of C1q, which is composed of six collagen-like structures connected to six globular head domains. C1q binds a C1r2C1s2 heterotetramer to form C1 complex. The depiction of the arrangement of subunits within C1 is based on the work of Ugurlar and colleagues [A) A schematic depiction of classical pathway complement activation is shown. C1q, the pattern recognition subunit of the C1 complex, binds to the targeted surface. C1q binding autoactivates the initiator serine protease, C1r, which then proteolytically cleaves C1s. Activated C1s cleaves complement proteins C2 and C4 leading to the surface formation of C4b2a, the CP /LP (Lectin Pathway) C3 convertase. C3 convertases then cleave C3 into C3a and C3b leading to CP/LP C5 convertase formation (C4b2a3b). Cleavage of C5 by C5 convertases releases the anaphylatoxin C5a and leads the formation of the membrane attack complex (C5b-9) on the surface of the target cell. The membrane attack complex is a lytic pore structure that can directly kill the targeted cell(s). For lleagues . BBK32-C(TIF)Click here for additional data file.S2 Figbbk32 orthologues bad16 and bgd19 from B. afzelii and B. garinii, respectively, were constructed using the pBBE22luc vector backbone. The resulting constructs were transformed into B. burgdorferi strain B314. B) PCR confirmation of bgd19 from B314/pBGD19, bbk32 from B314/pCD100, and bad16 from B314/pBAD16. All constructs contained the bad16, bbk32, bgd19 expressed from their native promoters. The Vector lane refers to the use of pBBE22luc as template for PCR with the oligonucleotide primers used to screen inserts. Values listed to the left indicate the size of markers in kilobases (kb). C) Quantitative RT-PCR shows that the expression of bbk32 orthologues  in strain B314 using their native promoters make transcripts equivalent or greater than B. burgdorferi sensu stricto bbk32. Expression of the bbk32 orthologues was compared relative to the constitutively expressed flaB gene . The qRT-PCR was done in triplicate and the mean values obtained for bbk32 was used as a comparator for the other orthologous genes .A) Schematic showing how the (TIF)Click here for additional data file.S3 Figsensu lato isolates tested. Antisera against BGD19 from B. garinii, BBK32 from B. burgdorferi, and BAD16 from B. afzelii were tested in immunoblots of protein lysates from B. burgdorferi strain B314 containing the vector pBBE22luc (B314/luc), as well as B314 strain expressing B. garinii bgd19 (B314/pBGD19), B. burgdorferi bbk32 (B314/pCD100), and B. afzelii bad16 (B314/pBAD16). Individual membranes were then probed with rat polyclonal antisera against BGD19-C, BBK32-C, and BAD16-C as specified on the right. In all instances, the reagent used recognized its homologous target protein best but also showed significant reactivity to the other heterologous proteins. Markers in kDa are indicated on the left. B) The BBK32 orthologues encoded by B. afzelii and B. garinii, designated as BAD16 and BGD19, respectively, are surface exposed in the surrogate B. burgdorferi B314 strain. B314/pBAD16 and B314/pBGD19, encoding BAD16 and BGD19, respectively, were grown, washed, and then either resuspended with Proteinase K  or buffer alone (denoted with a “-“). Following processing, the resulting samples were subjected to SDS-PAGE and immunoblotted with antiserum directed against either BAD16-C, the outer membrane P66 protein, or the subsurface FlaB protein. Given the cross-reactivity of anti-BAD16 with all B. burgdorferi BBK32 orthologues (panel A), the fate of BGD19 could be assessed with the anti-BAD16-C reagent. Protein markers are indicted in the left (in kDa).A) Antisera to BBK32 orthologues is cross reactive against all (TIF)Click here for additional data file.S4 Fig(206–348), which lacks six C-terminal residues relative to BBK32-C (i.e. BBK32206-354), retains high affinity C1r interaction and complement inhibitory properties. D) 2Fo-Fc density contoured at 1.2 σ for the entire BBK32 polypeptide.A-C) The construct used for crystallization, BBK32(TIF)Click here for additional data file.S5 FigSWISS-MODEL was used to produce homology models of A) BAD16-C and B) BGD19-C that are based on the crystal structure of BBK32-C (PDB: 6N1L). Residues that are non-identical between BAD16-C and BBK32-C are shown in red on the protein surface (panel A), while residues that differ between BGD19-C and BBK32-C are shown in yellow (panel B). C) The homology models of BAD16-C and BGD19-C are structurally aligned. The coloring scheme shown in panels A/B is retained except overlapping residues are now colored in orange. Surfaces that remain yellow represent residues that are uniquely different in BGD19-C relative to BAD16-C. D) Three of these residues were selected for the BXK32-C chimera protein used in this study including residue positions 308, 319, and 324 (BBK32 numbering). A SWISS-MODEL homology model of the BXK32-C chimeric protein, also based on the BBK32-C crystal structure, predicts these three residues would remain solvent exposed. Global Model Quality Estimation (GMQE) is used by SWISS-MODEL to provide an estimate of model accuracy. Values range between 0 and 1, with higher numbers indicating higher model reliability and are as follows: BAD16-C (GMQE = 0.81); BGD19-C (GMQE = 0.93); BXK32-C (GMQE = 0.97).(TIF)Click here for additional data file.S1 TableThe calculated equilibrium dissociation constants, rate constants, and associated fitting statistics are provided for surface plasmon resonance binding experiments.(DOCX)Click here for additional data file.S2 TableThe calculated half maximal inhibitory concentration (IC50) values and associated fitting statistics are provided for each experimental set of complement functional assays.(DOCX)Click here for additional data file.


IL-10. The results suggest that the calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect.High expression levels of the calcium-binding proteins S100A8 and S100A9 in myeloid cells in kidney transplant rejections are associated with a favorable outcome. Here we investigated the myeloid cell subset expressing these molecules, and their function in inflammatory reactions. Different monocyte subsets were sorted from buffy coats of healthy donors and investigated for S100A8 and S100A9 expression. To characterize S100A9high and S100A9low subsets within the CD14+ classical monocyte subset, intracellular S100A9 staining was combined with flow cytometry (FACS) and qPCR profiling. Furthermore, S100A8 and S100A9 were overexpressed by transfection in primary monocyte-derived macrophages and the THP-1 macrophage cell line to investigate the functional relevance. Expression of S100A8 and S100A9 was primarily found in classical monocytes and to a much lower extent in intermediate and non-classical monocytes. All S100A9+ cells expressed human leukocyte antigen—antigen D related (HLA-DR) on their surface. A small population (<3%) of CD14+ CD11b+ CD33+ HLA-DR− cells, characterized as myeloid derived suppressor cells (MDSCs), also expressed S100A9 to high extent. Overexpression of S100A8 and S00A9 in macrophages led to enhanced extracellular reactive oxygen species (ROS) production, as well as elevated mRNA expression of anti-inflammatory  The S100 calcium-binding proteins A8 and A9 (S100A8 and S100A9), also known as migration inhibitory factor-related proteins 8 (MRP8) and 14 (MRP14), are abundantly expressed in myeloid cells, such as circulating monocytes and neutrophils. Their level of expression can be used as a biomarker of inflammation in bacterial infections and autoimmune diseases ,3,4,5. AIn contrast, several reports have provided evidence that S100A8 and S100A9 can exert anti-inflammatory effects: this was shown in lipopolysaccharides (LPS)-induced endotoxemia and autoimmune myocarditis ,12. IntrS100A8 and S100A9 have a more favorable long-term outcome than rejections with low expression [S100A9 expression with the expression of CD11b and CD33 [The role of myeloid cells in allograft rejection has been increasingly appreciated ,18,19. Wpression ,21, suggand CD33 . MDSCs hand CD33 ,24. FurtBased on previous findings we hypothesize that S100A9+ myeloid cells have distinct immune regulatory properties. In the current study, we phenotypically characterized monocytes that differentially expressed S100A8 and S100A9, and identified a functional role of these calcium-binding proteins in macrophages.CD14 and CD16 in the three sorted populations verified the sorting strategy , intermediate (CD14+ CD16+), and non-classical (CD14− CD16+) monocytes A. Messenstrategy B. Expresstrategy B, which strategy A. Proteistrategy C,D. The strategy D. The reNext, we tested whether there is variation in S100A9 expression within the CD14+ monocyte population. For this, we subjected CD14+ enriched cells to cytospin analysis of S100A9 protein. The fluorescence intensity varied greatly between cells E. SimilaTo investigate whether S100A9-positive monocytes express HLA-DR on their surface and are potentially able to interact with CD4+ T cells, we analyzed S100A9 and HLA-DR expression by FACS on PBMC from healthy donors. HLA-DR and S100A9 were co-expressed in CD14+ monocytes A. HLA-DRWe further observed that MDSCs, which are characterized as CD14+ CD11b+ CD33+ HLA-DR− and constitute only a small percentage of the total monocyte population D, showedTo test whether S100A9high and S100A9low monocytes differ phenotypically in their cytokine expression profile, we combined intracellular S100A9 FACS staining with mRNA analysis of sorted cell populations. Since fixation and intracellular FACS staining disturbs the RNA quality inside the cells, the assay needed to be optimized first. Processed (fixed and intracellular stained) cells showed significantly reduced qPCR signals  compared to live cells A. ApplicTNFα, IL-1β, and of anti-inflammatory cytokines TGFβ1 and IL-10 -containing plasmid was transfected for 24 h. The high proportion of GFP-positive cells indicates a high transfection efficiency C. We didCalcium-binding proteins S100A8 and S100A9 are involved in various inflammatory disorders. Relatively high levels of S100A8 and S100A9 in kidney biopsies have a beneficial effect on long-term graft outcome, independent of the extent of myeloid cell infiltration. In this study, we phenotypically characterized the monocyte subsets that differentially express S100A8 and S100A9. We further showed that overexpression of S100A8 and S100A9 in macrophages leads to enhanced ROS production and IL-10 expression.S100A8 and S100A9 were abundantly expressed in the classical monocyte subset [Monocytes have been documented to consist of three main cell populations based on the expression level of CD14 and CD16: classical, intermediate, and nonclassical ,26,27. Ae subset ,29. At tAs the expression of S100A9 in the CD14+ HLA-DR-low subset was even slightly higher than that in the HLA-DR-high subset, it is clear that HLA-DR is not an appropriate cell surface marker for sorting monocytes that differently express S100A9 protein. Consistent with our earlier findings in transplant tissue , S100A9 S100A9 mRNA expression in the FACS S100A9high subset compared to the S100A9low subset, which gave confidence with respect to the reliability of the results for the other transcripts studied.We investigated S100A9high and S100A9low monocytes, based on intracellular FACS staining, for cytokine expression. Recently, a method for the isolation of high quality RNA from cells following fixation, intracellular staining, and FACS sorting by the use of buffers containing RNase inhibitors has been described . HoweverS100A9 expression levels were as high as the reference gene (GAPDH) and were certainly above the SmartFlare detection limit (Cq < 34). Consistent with our findings, Maria and colleagues demonstrated that SmartFlare fluorescence intensity does not correlate with the level of target transcript, but depends on the efficiency of probe accumulation [SmartFlare probes have been suggested as suitable tools to detect mRNA in single living cells. Several studies showed successful detection of mRNA using SmartFlare or NanoFlare probes ,33,34,35mulation . We founmulation . Mirkin mulation . This maIL-10, but not of pro-inflammatory cytokines TNFα and IL-1β. Despite the consistent IL-10 mRNA increase in all experiments, a statistically significant difference was not obtained between cells transfected with S100A8/A9 and those transfected with empty vectors, probably because of the variation in the range of increase between experiments. Despite our efforts using protein screening in the supernatant at 24 h after transfection, IL-10 protein could not be detected. Hence, it cannot be stated with certainty that IL-10 is a significant mediator in the induction of immune regulation. Especially in the light of the presence of HLA-DR on S100A9-positive cells, both ROS and IL-10 may represent parts of a pathway through which the S100 molecules trigger the induction of regulatory T cells and inhibition of effector T cells, thereby limiting or preventing a detrimental immune response to the graft.To investigate the functional effect of S100A8 and S100A9, we overexpressed both molecules in primary monocyte-derived macrophages. We did observe that this led to significantly increased ROS production, which was similar to what was found in our previous study using the established, secondary THP-1 macrophage cell line . ThroughIn summary, the results suggest that calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect, which may explain the previously observed beneficial effect of the local S100A8 and S100A9 expression on kidney graft survival ,21.The following reagents were used for the study: phosphate-buffered saline  pH 7.4 , distilled water , paraformaldehyde (PFA) , Saponin , recombinant ribonuclease inhibitor , bovine serum albumin (BSA) fraction V-molecular biology grade , PE mouse anti-human S100A9 Clone 1H9 , rabbit-anti-human S100A9 , goat-anti-rabbit IgG-AF488 , RPMI1640 , fetal calf serum (FCS), penicillin, streptomycin, L-glutamine , diphenyleneiodonium , GM-CSF , mouse anti-human antibody against CD14, CD16, and HLA-DR .Written informed consent was obtained from donors for use of buffy coats for scientific purposes. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation from buffy coats of anonymous healthy donors . CD14+ monocytes were isolated from PBMC by positive selection using human CD14 microbeads  according to the manufacturer’s instructions.Purified PBMC were stained with antibodies against CD14 and CD16 in 100 μL of FACS buffer. After washing twice, three monocyte subsets were sorted using FACSAria flow cytometer according to the CD14 and CD16 expression level. Sorted subsets were pelleted and lysed for RNA isolation.6 cells/mL. Three drops of cell suspension were transferred onto a glass slide using a cytospin 4 cytocentrifuge  at room temperature. Cells were fixed using cold acetone for 10 min and washed three times. Cells were incubated with a 1000-times-diluted rabbit-anti-human S100A9 antibody for 1 h at room temperature. After washing, slides were incubated with 1:200 diluted goat-anti-rabbit IgG-AF488 for 30 min. Images were acquired on a fluorescence microscope  using digital imaging software .CD14 microbeads-enriched monocytes (positive selection) were washed with PBS and resuspended at a concentration of 0.5 × 10Intracellular staining procedures, followed by RNA extraction and qPCR, were mainly based on a previous study . In brieg for 5 min at 4 °C and the supernatant was removed. Total RNA from intracellular stained cells was isolated using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE , by incubating with 100 μL of protease digestion buffer for 3 h at 50 °C. The other steps in the RNA isolation procedure were carried out according to the manufacturer’s instructions. When working with unstained, intact cells, total RNA was extracted using the NucleoSpin miRNA kit . Complementary DNA synthesis was performed as described previously [After flow cytometric sorting, the cell suspension was centrifuged at 3000eviously ,42.Primer sequences for quantitative PCR are provided in 6 per well in 6-well plates. After 5 days, transfection mixture containing 20 μL of Lipofectamine 2000 (Thermo Fisher) and 7.5 μg plasmid was added dropwise to the wells. After 5–6 h the medium was replaced by fresh complete roswell park memorial institute (RPMI) medium and incubated for another 24 h. The extracellular ROS production by the cells was measured as described previously [To allow differentiation into macrophages, CD14+ monocytes were cultured in the presence of 5 ng/mL GM-CSF for 5 days. Monocyte-derived macrophages were seeded at 1.5 × 10eviously .The THP-1 macrophage cell line was transfected with similar amounts of plasmid as described above, and incubated for 24 h. Cells were washed and resuspended for incubation for another 24 h in complete medium, either containing no stimulants or containing 100 ng/mL LPS. The cells were then pelleted and lysed for RNA isolation.t-test, and the difference between the two groups was calculated by one-way ANOVA with Tukey’s multiple comparison tests.Statistical analyses were performed using SPSS statistics 23 and GraphPad Prism 7.02. All experiments were performed at least three times. The statistical difference between the two groups was calculated by


Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of  TARGET OF FLC AND SVP1 (TFS1) as a novel target of FLC and SVP, and demonstrate that TFS1 contributes to proper flowering-time control. Moreover, we provide a detailed mechanistic view of how TFS1 transcription is controlled during reproductive development through the repressive activity of FLC/SVP being overcome by the transcriptional activator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. Thus we further elucidate the network of genes repressed by FLC/SVP to block flowering and determine mechanisms by which their repressive activity is overcome during the initiation of flowering.The initiation of flowering in plants is exquisitely sensitive to environmental signals, ensuring that reproduction occurs at the appropriate time of year. The sensitivity of these responses depends upon strong repression of flowering under inappropriate conditions. FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) are related transcription factors that act in concert to strongly inhibit flowering in crucifer plants through repressing transcription of their target genes. Many direct FLC/ SVP targets have been identified in genome-wide studies, however few of these genes have been characterized for their roles in regulating flowering time or other aspects of reproductive development. Here, we characterize  Arabidopsis thaliana, the MADS-box transcription factor FLOWERING LOCUS C (FLC) plays a major role in this network as an inhibitor of floral transition [FLC is repressed by extended exposure to cold that mimics winter conditions  so that flowering can proceed when plants are subsequently exposed to warm in spring. The repression of FLC transcription by accumulation of histone modifications in response to cold has been extensively studied [TARGET of FLC and SVP 1 (TFS1). We show that this gene acts in the network downstream of FLC and other floral regulators, and has an important role on the flanks of the meristem during the early stages of floral development.The transition from vegetative to reproductive development in plants is controlled by a complex transcriptional network that responds both to environmental cues and endogenous hormonal signals , 2. In Aansition , 4. Tran studied  and FLC  studied –8. Never studied , 9–12 toFLOWERING LOCUS T (FT), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SEPALLATA 3 (SEP3). MADS box transcription factors are proposed to bind DNA as dimers and tetramers [SVP are early flowering and exhibit increased levels of SOC1 and FT mRNAs [FLOWERING LOCUS T (FT) in leaf veins to repress its transcription [FT chromatin, most probably through the activity of the histone methyltransferase CURLY LEAF (CLF) and the H3K27me3-associated protein LIKE HETEROCHROMATIN PROTEIN1 (LHP1) [FT [FLC binds to several hundred target genes, but only a small subset of these are conserved between species . Genes ietramers , 14, andetramers , 10. MutFT mRNAs . How FLCFT mRNAs , 18, to cription . Moreove1 (LHP1) . AdditioHP1) [FT , 21.FRUITFULL (FUL) and MIR172b to activate the basal floral promotion pathway under non-inductive environmental conditions [FUL and MIR172b to orchestrate the removal of the H3K27me3 mark and activate transcription [trans-activation activity and contribute to the induction of expression of the floral meristem identity gene APETALA1 (AP1) in the low gibberellin context present in the cells that give rise to the floral primordium [Other targets of FLC and SVP that are involved in floral induction encode the MADS-box transcription factor SOC1 and the plant-specific transcription factor SPL15 –27. Bothnditions , 22, 28.imordium .TFS1, which encodes a B3-type transcription factor that is a member of the REPRODUCTIVE MERISTEM (REM) family [TFS1 expression is induced at the periphery of the SAM by SOC1 and the age-regulated transcription factor SPL9 through coordinated recruitment of the histone demethylase REF6 and the chromatin remodeler BRAHMA (BRM) [Here, we show that ) family , 31, conMA (BRM) –34. ThisTFS1 is part of a gene family including REDUCED VERNALIZATION RESPONSE1 (VRN1), VERDANDI (VDD) and VALKYRIE (VAL), and that REM18 and REM19 are the closest homologs of TFS1 within a sub-branch of the phylogenetic tree  analysis was performed using chromatin extracted from the aerial parts of 15-day old plants grown under inductive long days (LDs) and using antibodies that were raised against FLC [TFS1 was detected after ChIP of FLC or SVP  to inductive LDs , and by showing that a second allele (tfs1-2) caused a similar late-flowering phenotype under LDs  2 (PRC2) , 19. PRC H3K4me3 . H3K27me mutants . However of TFS1 , S3A Fig of TFS1  was usedH3K27me3 . TherefoTFS1 in the Col-FRI flc-3, Col-FRI svp-41 and Col-FRI flc-3 svp-41 mutants, whereas it was not present at the gene in Col-FRI plants  is present at genes that are actively transcribed . EnrichmI plants . These dcurly leaf (clf), which impairs the activity of an enzyme that catalyses H3K27me3 deposition [Col-FRI plants [Col-FRI clf-2 mutant plants expressed highly elevated levels of TFS1 mRNA in the apex, while still expressing high levels of FLC and SVP mRNA  , 43, 44, and FLC , suggestild-type . By cont mutants . ConsistP or FLC . In suppH3K27me3 , reducedaffected , S3H FigTFS1 stop codon  gene fusion was constructed that contained the entire intergenic region flanking TFS1 on the 5’ and 3’ sides  digestion followed by tiled oligo qPCR was employed to identify well-positioned nucleosomes near the SOC1, REF6 and BRM bound site at the 3’ end of TFS1. The MNase-qPCR analysis identified in ref6-1 and brm-1 mutants a nucleosome at a position that encompasses the binding site for SOC1 and this nucleosome was destabilized in Col (TFS1 through recruitment of REF6 and BRM.To test whether BRM recruitment leads to activation of d in Col . TherefoTFS1 appeared similar to those of SPL9 [APETALA1 (AP1) to regulate floral fate [TFS1. To test for the molecular effect of SPL9, RNA was extracted from apices of SPL9::GFP:rSPL9 to monitor TFS1 transcript abundance by RT-qPCR. TFS1 transcript levels were strongly increased in SPL9::GFP:rSPL9 plants compared to wild-type  transgenic lines  was employed to test for DNA loop formation in Col and 35S::miR156b, in which several redundant SPL transcription factors are reduced in expression [TFS1  was detected in protein extracts from shoot apical tissue of ic lines . The coopression  leading on [TFS1 . The 3C ner Figs . TogetheTFS1 in a SOC1 dependent-manner to increase chromatin accessibility and TFS1 transcription , AP1 and SEPALLATA3 (SEP3) was also reduced in this genotype, although SPL9 protein level was not affected , which is in a Landsberg erecta (Ler-0) genetic background. Seeds were sown on soil or on full-strength Murashige and Skoog (MS) medium containing 1% sucrose, stratified for 3 days at 4°C, and grown at 22°C under either long-days  or short-days . Plant age was measured when seeds were transferred from stratifying to ambient growth conditions.All seed stocks are in the Columbia-0 (Col-0) genetic background and were obtained from the Nottingham re NASC;  except fTFS1 genomic region was cloned by PCR with Phusion Enzyme (New England Biolabs) according to the manufacturer’s recommendations and used to generate TFS1::TFS1::9xAla-Venus. To introduce 9xAla-Venus coding sequence, we employed Polymerase Incomplete Primer Extension (PIPE) cloning method [Agrobacterium to transform Col plants by floral dip [Full-length g method  and plasoral dip . The seqμg RNA was reverse transcribed with an oligo(dT) primer, RNAseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific) and SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). The cDNA equivalent of 20ng of total RNA was used in a 12 μL qPCR reaction on a Roche Light Cycler 480 instrument (Roche) with either iQ SYBR Green Supermix (BioRad) or GoTaq qPCR Master Mix (Promega) and quantified using the UBC21 (AT5G25760) as a reference gene to which data was normalized [Total RNA of indicated genotypes at different days after sowing from leaves and apices was isolated with NucleoSpin RNA plant kit (Macherey-Nagel). DNA was removed by an on-column treatment with rDNase and 2 rmalized . The meaμM was used to introduce protein-protein crosslinks prior to formaldehyde-assisted protein-chromatin crosslinking. To determine fold enrichment levels, ChIP-DNA was quantified on a Roche Light Cycler 480 instrument (Roche) with iQ SYBR Green Supermix (BioRad) and normalized against ACT8 (AT1G49240). In ChIP assays in which histone modifications were tested, the values of the tested histone marks were normalized against histone H3. The average of three biological replicates is shown and list of primers used for fold enrichment analyses can be found in ChIP was performed as previously described with minor modifications . In brie3C assay was performed as described previously with minor modifications. A total of 2g of above-ground tissue of 15 day-old LD-grown plants was used for 3C study. Chromatin DNA was digested for 16 hrs at 37°C with 400U Sau3AI .All image processing and figure construction was performed with Photoshop  Increased expression of TFS1 in apices of Col-FRI plants when transferred to ambient growth condition after vernalisation. NV, non-vernalised; V, weeks vernalised in short-days (SD). E) Spatial pattern of expression of TFS1 assessed by in-situ hybridization after floral transition. DAS, days after sowing.A) Phylogenetic tree for B3 type gene family based on ClustalW alignment with a bootstrap value of 1.000 replicates. B) Immunoblot to test for cross-reactivity of SVP. Triangle indicates specific whereas asterisks indicate non-specific bands. Values on the left of the Western-blots indicated molecular weight (kDa). C) Transcript level of (TIF)Click here for additional data file.S2 Figtfs1 mutant plants and Columbia grown in either LDs (A) or in SDs (C). Leaf number of tfs1 plants compared to Columbia grown in LDs (B) and in SDs (D). Statistical significance was calculated using Student’s t-test; **P < 0.01. E) Representative gene model for TFS1 and T-DNA insertion used in this study. F) Total leaf number of tfs1-1 and tfs1-2 plants compared to Col. Statistical significance was calculated using Student’s t-test; *P < 0.05. G) Inflorescence of Col-FRI flc svp tfs1 plants. H) Floral structures of Col-FR flc svp tfs1 plants. Particular features are marked: blue arrow, misplaced floral organs; white arrow, ectopic ovules; yellow arrow, stigmatic papillae on leaf-like structures; red arrow, leaf-like anthers.A to D) Comparison of (TIF)Click here for additional data file.S3 FigTFS1 in Col-FRI flc svp. B and C) H3K4me3 levels are increased in Col-FRI flc svp (B) and Col-FRI clf and Col-FRI jmj14 (C) plants. Values were scaled to set highest value in Col-FRI flc svp to 1. D) Reduced TFS1 transcript levels in Col-FRI plants suppressed by clf mutation. E and F) Transcript levels of FLC (E) and SVP (F) in leaves and apices of Col-FRI clf plants. Numbers at x-axis indicate number of long-days (LD) for which plants were grown prior to harvest. G) Reduced TFS1 transcript levels in Col-FRI plants suppressed by jmj14 mutation. H and I) Transcript levels of FLC (H) and SVP (I) in leaves and apices of Col-FRI jmj14 plants. Numbers at x-axis indicate number of long-days (LD) for which plants were grown prior to harvest. Statistical significance was calculated using Student’s t-test; *P < 0.05, **P < 0.01, n.s. P > 0.05.A) H3K27me3 levels are increased at TTS of (TIF)Click here for additional data file.S4 FigTFS1. B and C) Top: ChIP-qPCR analysis of SOC1 (B) and SVP (C) on CArG-boxes at TFS1 in 35S::SOC1:GR soc1 plants after DEX treatment. Bottom: Diagram of TFS1 locus with CArG-boxes and amplicons used for ChIP-qPCR. Red and orange triangles indicate CArG-boxes in TFS1. Green and red lines represent amplicons used for ChIP-qPCR study. D) ChIP-qPCR analysis of H3K27me3 at TFS1 in 35S::SOC1:GR soc1 plants after DEX treatment. B to D, values were scaled to set value of first primers at 20min after DEX treatment in 35S::SOC1:GR soc1 to 1. GB, indicates Gene Body amplicon used for ChIP-qPCR. Statistical significance was calculated against mock treatment using Student’s t-test; *P < 0.01, n.s. P > 0.05. Representative confocal images of independent transformants of E) TFS1:9AV, F) TFS1:9AV mCArGII and G) TFS1:9AV mCArGI+II. H) Abundance of TFS1 mRNA in indicated transgenic plants as well as in Col and tfs1 controls.A) SOC1:GR-induced transcriptional activation of (TIF)Click here for additional data file.S5 FigTFS1::TFS1:9AV in tfs1. C and D) Representative photograph (C) and total leaf number (D) of TFS1::TFS1:9AV mCArGII in tfs1. E and F) Representative photograph (E) and total leaf number (F) of TFS1::TFS1:9AV mCArGII in soc1. G and H) Representative photograph (G) and total leaf number (H) of TFS1::TFS1:9AV mCArGI+II in tfs1. In each case, transformants are shown in the same order in the representative photograph as in the leaf number plot. Statistical significance was calculated using Student’s t-test; *P < 0.1, **P < 0.05, ***P < 0.01, n.s. P > 0.05.A and B) Representative photograph (A) and total leaf number (B) of (TIF)Click here for additional data file.S6 FigTFS1:9xAla-Venus (yellow) expression at the apical meristem. DAPI (blue) serves to highlight individual cells. B) Side view of TFS1:9xAla-Venus (yellow) localisation at the shoot apical meristem. C to G) TFS1:9xAla-Venus localises to the nucleus of sepal cells (C), in young flowers and at the base of pedicels (D) and in flowers of stage stage 3 (E), stage 2 (F) and stage 4 (G) after LMB treatment.A) Top view of (TIF)Click here for additional data file.S7 FigTFS1 locus. Statistical significance was calculated against the respective amplicon of 35S::YFP:GFP. B) ChIP-qPCR for temporal binding of SPL9:GR at TFS1 after DEX treatment. Statistical significance was calculated against the 20min DEX treatment. C to E) ChIP-qPCR for MED18 (C), RNAPII (D) and H3K4me3 (E) at TFS1 locus. Plants were grown for 15 LD and harvested at ZT8. F) Spatial pattern of expression of TFS1 assessed by in-situ hybridization during floral transition in Col and GFP:rSPL9. Plants were grown for 2 weeks in SD and then transferred to permissive LD. G) Confocal images of TFS1:9AV mGTACa1/a2 mutant transgenic lines. Statistical significance was calculated using Student’s t-test; *P < 0.05, **P < 0.01, n.s. P > 0.05.A) ChIP-qPCR for rSPL9 binding to putative SPL-binding sites at (TIF)Click here for additional data file.S8 FigTFS1 transcript abundance in short-days is increased by SPL and SOC1 (A) and REF6 (B). Data are shown for 3–8 weeks after germination and values were scaled to set value of 3 weeks SD-grown Col plants to 1. C) Reduced expression of TFS1 in ga1 under LD conditions. D) Spatio-temporal expression pattern of TFS1 assessed by in situ hybridization in LD. E) TFS1 mRNA levels are reduced in apices of spl mutants during floral transition. Values were scaled to set value of 7 LD of Col to 1. F to H) Reduced RNAPII (F) and H3K4me3 (G) and increased H3K27me3 (H) levels at TFS1 in spl mutants. Values were scaled to highest value in Col (F and G) and spl9 spl15 (H) to 1. In A to H, Plants were harvested at ZT8.A and B) (TIF)Click here for additional data file.S9 FigTFS1 depend on SOC1 (A), BRM (B) and SPL (C). D to F) H2A.Z incorporation at TFS1 depends on SOC1 (D) and BRM (E) but not on SPL (F). ChIP-PCR was performed on 15 LD grown plants that were harvested at ZT8 (A to F).A to C) WDR5 levels at (TIF)Click here for additional data file.S10 FigGFP:rSPL9 brm plants display altered plant architecture (A) and partial transformation of flowers into carpelloid structures (B). C) GFP Western analysis to detect GFP:SPL9 in Col and brm. Values on the left of the Western-blots indicated molecular weight (kDa). D and E) Comparative expression analysis of marker genes expressed in leaves (D) and apices (E) of Col, GFP:rSPL9 and GFP:rSPL9 brm plants. X-axis indicates number of long-days (LD) for which plants were grown prior to harvest.A and B) 5-weeks old long-day grown (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy.Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple clinical trials. However,  Pathogens often find ways to turn down cell-intrinsic antipathogen immune responses by the host. Similarly, cancer cells use various mechanisms to evade attack by immune cells. One of the common mechanisms is suppression of the stimulator of interferon genes (STING)-dependent innate immune response. Using potent and specific small-molecule inhibitors and genetic-depletion approaches, we found that the silenced STING pathway can be reactivated in breast cancer cells by suppressing KDM5 demethylases. Activation of the STING pathway led to a robust interferon response, which blocked viral infection, and was associated with increased tumor-infiltrated lymphocytes and better patient survival in multiple cancer types. This discovery has major clinical implications for treating both pathogen infection and cancer because KDM5 inhibition provides a fast, robust, and reversible control of innate immune response. Since the discovery of histone demethylase activity of KDM5 proteins a decade ago, significant efforts have been dedicated to developing KDM5 inhibitors for clinical applications. In fact, a KDM5 inhibitor recently entered phase I clinical trial for treatment of hepatitis B infection. Here, we provide mechanistic insights on how KDM5 inhibitors block viral infection. Moreover, our results suggest that KDM5 inhibitors can also be combined with other cancer immunotherapies. Evasion from immunosurveillance by cancer cells is a major cancer hallmark , and resPattern recognition receptors (PRR) are cell surface and intracellular sensors that recognize pathogen-associated and abnormal-self molecular patterns, e.g., nucleic acids, and trigger intracellular signaling cascades to activate cell-intrinsic antipathogen or antitumor responses . Cyclic KDM5B is up-regulated in breast cancer cells overexpressing the ERBB2/HER2 oncogene , 0%–20% glycerol, and 25 mM [Na/K] dibasic/monobasic phosphate) and CPI-48 (approximately 500 μM), allowing the crystal to remain in this drop overnight for CPI-48 to exchange with αKG. The crystals were then mounted into nylon cryoloops  and frozen in liquid nitrogen after the addition of more glycerol  to the mother liquor as a cryoprotectant. X-ray diffraction data were collected SER-CAT beam-line 22-ID at the Advanced Photon Source at Argonne National Laboratory at 100 K with 1-degree oscillation images, and the structure was determined by molecular replacement and refinement performed as described , and plasmid transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The sequence of dsDNA90 was described previously , transfeHistone extraction was conducted as described previously .https://chopchop.rc.fas.harvard.edu/) and cloned into LentiCRISPRv2. Knockout cells were generated as described previously pyrimidin-4(1H)-one moiety (PDB 5FPL) (D) [50 values (mean +/− SD) (lower panel) of all 4 compounds against KDM5B (1–755) ΔAP from 3 independent experiments. (H) AlphaLISA assays showing that Dong-A-167 is a potent inhibitor of KDM5A, KDM5B, and KDM5C. Shown were representative dose response curves (upper panel) and IC50 values (mean +/− SD) (lower panel) of Dong-A-167 against KDM5 demethylases from 3 independent experiments. The numerical values used to generate graphs in panel G and H are available in 50, half maximal inhibitory concentration.(A) KDM5-C70 was designed as a cell-permeable prodrug that is hydrolyzed by intracellular esterase(s) to generate KDM5-C49, which contains an isonicotinic acid moiety with a carboxylic acid (PCT WO 2014053491) , 32. (B)FPL) (D) . (E) GDCFPL) (D) . (F) Com(TIF)Click here for additional data file.S2 Fig#p < 0.01 for inhibitors versus DMSO (panel C–E), for KDM5 siRNA versus average of 2 control siRNAs (panel G). The numerical values used to generate graphs in panel C–E and G are available in p-value; RT-qPCR, RT-qPCR, reverse transcription followed by quantitative PCR; siRNA, small interfering RNA.(A) Western blot analyses of MCF7 cells treated with 1 μM KDM5-C70 for 3 days. (B) Pathways that were up-regulated by 3 μM KDM5-C70 or CPI-48 treatment, revealed by GSEA. The gene set database of h. all. v6.1. symbols. gmt  was used. All the up-regulated pathways in inhibitor-treated cells with FDR q value < 0.05 were shown. (C) RT-qPCR analysis of MCF7 cells treated with 1 μM KDM5-C70, 10 μM each of Dong-A-167, GDC-50, or CPI-48 for 6 days.  RT-qPCR analysis of SKBR3 cells treated with 5 μM KDM5-C70 (panel D) and BT474 cells treated with 1 μM KDM5-C70 (panel E) for 6 days.  Western blot (panel F) and RT-qPCR (panel G) analyses of MCF7 cells 5 days after transfection with the indicated siRNAs. KDM5BC, 2 siRNAs targeting KDM5B and KDM5C. Representative data from triplicate experiments are shown. Error bar denotes SEM. (TIF)Click here for additional data file.S3 Fig#p < 0.01 for inhibitors versus DMSO (panel B and E); KDM5-C70 medium versus mock medium (panel G). ^p < 0.01 for knockout sgRNA versus control sgRNA (panel E). The numerical values used to generate graphs in panel B and E–G are available in (A) T7 endonuclease assays showing the genome editing efficiency of MCF7 cells with CRISPR/Cas9-mediated TLR3 knockout.  RT-qPCR (panel B) and western blot (panel C) analyses of control or IRF3 knockout MCF7 cells generated by 3 independent sgRNAs against IRF3  after treatment with DMSO or 1 μM KDM5-C70 for 6 days. sgRNA IRF3-1 was used in (TIF)Click here for additional data file.S4 Fig#p < 0.01 for inhibitors versus DMSO (panel B and C). The numerical values used to generate graphs in panel A–C are available in (A) Flow cytometry plots (left panel) and quantification of GFP-positive cells (right panel) in MCF7 cells with knockout of the indicated genes 24 hours after infection with VSV-GFP at MOI 0.5. Cells were pretreated with DMSO or 1 μM KDM5-C70 for 5 days, followed by no treatment for 1 day before viral infection. (B) Representative images (left panel) and quantification of relative intensity (right panel) of control or IRF3 knockout MCF7 cells 3 days after infection with vaccinia viruses at MOI 0.25. MCF7 cells were pretreated with DMSO or 1 μM KDM5-C70 for 5 days, followed by no treatment for 1 day before viral infection. (C) qPCR analysis of DNA copy number of vaccinia viruses in growth media from the cells in panel B. Representative data from triplicate experiments are shown in panel C. Three biological replicates are shown in panel A and B. Error bar denotes SEM. (TIF)Click here for additional data file.S5 Fig(A–D) Western blot analysis of the indicated cell lines after treatment with DMSO or 1 μM KDM5-C70 for 6 days.  RT-qPCR (panel E) and western blot (panel F) analyses of control or KDM5B/KDM5C double KO MCF7 cells. (G) RT-qPCR analysis of MCF7 cells treated with control or KDM5B/KDM5C siRNAs. (H) Western blot analysis of control or IRF3 KO MCF7 cells 5 days after transfection with the indicated siRNAs. Representative data from triplicate experiments are shown. Error bar denotes SEM. The numerical values used to generate graphs in panel E and G are available in (TIF)Click here for additional data file.S6 FigSTING genomic region in K562 cells  and KDM5C in ZR-75-30 cells  [STING, but not cGAS and downstream genes OAS2, IFI44L, and IFI44. #p < 0.01 for the comparisons shown in panel A and B inhibitors versus DMSO. The numerical values used to generate graphs in panel A and B are available in  H3K4me3 ChIP-qPCR analysis of MCF7 cells (panel A) or BT474 cells (panel B) treated with DMSO or 1 μM KDM5-C70 for 1 day. (C) Analysis of ChIP-seq data for KDM5B binding at the r panel) . Heat ma(TIF)Click here for additional data file.S7 Fig RT-qPCR analysis of MCF7 cells with the indicated treatment. MCF7 cells were treated with 10 μM VE821 for 3 days (panel A) or 1 μM KDM5-C70 for 4 days (panel B), followed by 1-day treatment with 0.2 μM LMB. (C) dsDNA and DAPI staining of MCF7 cells treated with DMSO or 1 μM KDM5-C70 for 3 days. Surface plots of Z-stack images generated with Huygens. Scale bar, 10 μm. (D) Western blot analysis of SKBR3 cells with knockout of the indicated genes. The numerical values used to generate graphs in panel A and B are available in (TIF)Click here for additional data file.S8 Fign = 302. (B) Correlation between KDM5B and CXCL10 or STING and CXCL10 in TCGA CESC. (C) Correlation between KDM5B expression and CD8+ T-cell infiltration in TCGA CESC. CESC, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; STING, stimulator of interferon genes; TCGA, The Cancer Genome Atlas.(A) Correlation between KDM5B and STING in TCGA CESC. (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TablesgRNA, single guide RNA.(DOCX)Click here for additional data file.S3 TableRT-qPCR, reverse transcription followed by quantitative PCR.(DOCX)Click here for additional data file.S4 TableChIP-qPCR, chromatin immunoprecipitation followed by quantitative PCR.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.


To investigate the prevalence of common genetic variants that can serve as markers of thrombophilia and warfarin pharmacogenetics in Bosnia and Herzegovina.F5 c.1601G>A (factor V Leiden), F2 c.*97G>A (factor II or prothrombin mutation), F13A1 (factor XIII) c.103G>T, MTHFR (methylenetetrahydrofolate reductase) c.665C>T and c.1286A>C, as well as PAI-1 (plasminogen activator inhibitor 1) c.-816A>G and c.-844G>A as markers of thrombophilia risk, and *2 and *3 alleles of CYP2C9 (cytochrome P450 2C9) and five variants of VKORC1 (vitamin K epoxide reductase complex subunit 1) as markers of warfarin pharmacogenetics. DNA was isolated from buccal swabs using salting out method, while genotyping was performed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.The study was performed between August and October 2017 on 130 healthy unrelated adult volunteers from Bosnian-Herzegovinian population sample. The prevalence of the following genetic variants was determined: F5 c.1601G>A and F2 c.*97G>A were 0.023 and 0.008, respectively. Combined data for the markers of warfarin pharmacogenetics imply that 57.4% study participants can be expected to metabolize warfarin at an extensive, 40.3% at intermediate, and 2.3% at a poor rate.Minor allele frequencies for two main thrombophilia risk factors, This study reports the first extensive population genetic data for thrombophilia and warfarin pharmacogenetic markers in Bosnia and Herzegovina. Allele frequencies of genetic variants are within the general average for European populations, and their presence implies the necessity of introduction of personalized medicine in warfarin-mediated antithrombotic therapy. F5 c.1601G>A , F2 c.*97G>A , two MTHFR (methylenetetrahydrofolate reductase) variants, two PAI-1 (plasminogen activator inhibitor 1) variants, and F13A1 (factor XIII) c.103G>T   .Warfarin-mediated anticoagulation is currently considered to be a risky clinical therapy due to numerous interactions and interpatient variations in mean daily warfarin dose and narrow drug therapeutic range ,12,13. WThe aim of this study was to analyze the frequency of mutant alleles in two abovementioned sets of markers in Bosnia and Herzegovina (B&H). Additionally, we aimed to propose a simple and affordable method for genotyping relevant genetic variants that enables successful and safe implementation of personalized warfarin treatment in routine clinical practice.Buccal swab samples were collected from 130 unrelated adult volunteers from B&H. Sample collection was performed in the medical laboratory Prolab  between August and October 2017. Laboratory users were asked to participate in the study if they had no history of thrombotic events. In order to achieve a balanced distribution of participants’ demographic characteristics we included participants of both sexes, different age groups, and originating from different parts of the country as assessed by the origin of their grandparents. The size of the study population was determined according to previous population studies conducted in B&H ,16 and wAll genotyping procedures were performed in Bioglobe Laboratory, Hamburg, Germany. In order to analyze thrombophilia markers and pharmacogenetic markers of warfarin metabolism, two multiplexes and one uniplex iPLEX assays were set-up as follows:F5 c.1601G>A, F2 c.*97G>A, MTHFR c.665C>T and c.1286A>C, PAI-1 c.-816A>G and c.-844G>A, and F13A1 c.103G>T;1. Multiplex reaction for seven thrombophilia markers, including VKORC1 (vitamin K epoxide reductase complex subunit 1) c.-1320G>A, VKORC1 c.-1639G>A, VKORC1 c.-679A>G, VKORC1 c.174-136C>T, VKORC1 c.283 + 124G>C and CYP2C9 (cytochrome P450 2C9) c.1075A>C (CYP2C9*3);2. Multiplex reaction for six markers of warfarin pharmacogenetics: CYP2C9 c.430C>T (CYP2C9*2).3. Uniplex reaction for the marker of warfarin metabolism Primer selection and plex design was performed in Assay Design Suite v2 . PCR reactions were set-up in 384-well plate with reaction components and thermal cycling conditions as previously described . In ordeIon exchange sample conditioning was performed to remove excess cations from the sample-containing wells using Clean Resin (Agena Bioscience) . 15-nL shttp://biology-assets.anu.edu.au/GenAlEx/Welcome.html) using χ2 statistics at the significance level of 0.05 (http://genepop.curtin.edu.au/) at the significance level of 0.01 (Testing for Hardy-Weinberg equilibrium (HWE) was performed in GenAIEx v. 6.5 . All study loci were in agreement with HWE . Using log likelihood ratio, two pairs of loci were found to be in strong linkage disequilibrium: 1) MTHFR c.665C>T and MTHFR c.1286A>C and 2) PAI-1 c.-816A>G and PAI-1 c.-844G>A (P < 0.01 in both cases).Allele frequencies for two most significant markers of inherited thrombophilia were 0.023 and 0.008 for ribution  , while age and sex were not significantly associated with variant allele incidence . Three analyzed polymorphic VKORC1 loci, c.-1639G>A, c.174-136C>T, and c.283 + 124G>C, were in complete linkage disequilibrium (P < 0.001). Statistical tests were employed only for the loci found to be polymorphic.Genotype data for markers of warfarin pharmacogenetics  revealedCYP2C9 c.430C>T and c.1075A>C and VKORC1 c.-1639G>A genotypes was performed  , joint aerformed . A totalThrombophilia risk markers and genetic markers of warfarin pharmacogenetics were detected in a considerable proportion of general Bosnian-Herzegovinian population. The calculated allele frequencies are close to the previously reported European average.F2 c.*97G>A (PTM) of 0.008 in the present research is in agreement with previous studies in B&H and worldwide  states that the informativeness of MTHFR testing is limited, and that homocysteine levels should be measured instead. In addition, ACMG recommends against MTHFR testing for the analysis of inherited thrombophilia, recurrent pregnancy loss, or for at-risk family members . Ma. MaMTHFRof 0.594 .F13A1 c.103G>T, also known as Val35Leu, has been assigned a protective role against venous thromboembolism, myocardial infarction >4. The same is true for haplotypes A (warfarin-sensitive) and B (warfarin-resistant), consisting of c.-1639G>A and four additional loci, including c.174-136C>T and c.283 + 124G>C, inherited together due to extremely high level of linkage disequilibrium , followed by Europeans (0.37) and Africans (0.10). In US Hispanic population, MAF for this marker was 0.46  was the first CYP2C9 variant discovered and it stays the most studied SNP on this gene. Arg144Cys substitution encoded by this SNP disables a proper interaction of cytochrome P450 with NADPH-dependent cytochrome P450 oxidoreductase and decreases the rate of enzyme activity. Another important variant, CYP2C9 c.1075A>C (CYP2C9*3), influences enzyme conformation, thus lowering its ability to bind different substrates, including warfarin  by genotyping 37 T2DM patients and 44 healthy controls, reported a low mutant allele frequency of 0.09) (ectively . In the ectively . The onlof 0.09) .Novel or direct oral anticoagulants  are presently considered as an important improvement to traditional oral anticoagulation therapy, since they are less prone to interpatient variation, have stable pharmacokinetic and pharmacodynamic profiles exhibiting less interactions, and do not require frequent INR monitoring, thus decreasing or abandoning the need for warfarin dosing algorithms ,49. EuroF2 c.*97G>A (PTM), VKORC1 c.-1639G>A, and CYP2C9 variants c.430C>T and c.1075A>C in a study group of warfarin-anticoagulated patients and a control group to assess their importance for personalization of antithrombotic treatment.The limitations of the present study include the limited non-representative sample size of 130 individuals from the general population. This study thus does not offer information on the risk of thromboembolic events or recommendations for warfarin dosing at the population level. It is also important to note that this study alone does not provide guidelines regarding the duration or intensity of warfarin therapy in Bosnian-Herzegovinian patients. Future research should study the prevalence of the major genetic variants, including FVL, This is the first study reporting general population data for a panel of 14 genetic variants of inherited thrombophilia and warfarin pharmacogenetics in B&H. The results show that these genetic variants are considerably present in the general population of B&H. The frequency of detected minor variants is in agreement with previously published data for European populations. Implications of this study are important in that it paves the way for an integrated analysis of patient’s unique genomic makeup to offer individualized prophylaxis and treatment of thrombotic events.