TLR9 signaling induces type I IFN (IFN-I), which enhances MHC-I Ag cross processing, but TLR2 sig... more TLR9 signaling induces type I IFN (IFN-I), which enhances MHC-I Ag cross processing, but TLR2 signaling does not. Moreover, we previously reported that TLR2 signaling by Mycobacterium tuberculosis (Mtb) or other TLR2 agonists inhibited TLR9 induction of IFN-I and IFN-I-dependent MHC-I Ag cross processing (Simmons, 2010, J. Immunol.). In the current studies, a TLR2 agonist inhibited IFN-I mRNA induction when added together with TLR9 agonist for 2 to 24 h. TLR2 agonist inhibited TLR9-induced first wave IFN-I, since the inhibition was manifested in IFN-I receptor knock out DCs, which lack the second wave positive feedback mechanism, whereas second wave IFN-I mRNA induction by exogenous IFN-I was not inhibited. TLR2 also inhibited IFN-I induced by TLR7, another MyD88-dependent IFN-inducing receptor, but did not inhibit IFN-I induction by TLR3 or TLR4 (both TRIF-dependent, MyD88-independent). The inhibitory effect of TLR2 was not dependent on new protein synthesis or intercellular signaling. Current studies are further addressing the intracellular mechanism whereby TLR2 inhibits induction of IFN-I. This novel mechanism, whereby TLR2 inhbits IFN-I induction by TLR7/9, may shape immune responses to microbes that express ligands for both TLR2 and TLR7/TLR9, or responses to bacteria/virus coinfection.
Objectives To report methods and findings of 2 autopsies with molecular evaluation of severe acut... more Objectives To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals. Methods Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks. Results Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation. Conclusions SARS-CoV-2 testing and viral sequencin...
Krüppel-like factor 4 (KLF4) is a transcription factor that polarizes macrophages towards an anti... more Krüppel-like factor 4 (KLF4) is a transcription factor that polarizes macrophages towards an anti-inflammatory (M2) phenotype, which suggests that it may regulate immune responses to Mycobacterium tuberculosis (Mtb), as immune evasion by Mtb may be due to an overabundance of anti-inflammatory mediators in response to infection. We hypothesize that myeloid KLF4 is permissive of immune evasion by Mtb, causing decreased control of Mtb infection. There are no prior in vivo data on the role of KLF4 in infection. Using mice with myeloid-specific knockout of KLF4 (LysMCre/CreKLF4fl/fl, abbreviated Mye-KO) we observed decreased Mtb CFU in macrophage cultures in vitro and in lungs of Mye-KO mice early in infection (day 14) relative to wild-type controls. However, these experiments also revealed complexities to the KLF4-Mtb relationship. Despite their improved control of early infection, at later time points Mye-KO mice developed worsened clinical disease features, such as wasting, which suggests that the loss of KLF4 results in pathologic immune state. In addition, we found discrepancy between KLF4 RNA and protein expression during Mtb infection, suggesting that KLF4 is regulated at a posttranslational level during infection. We are designing an in vitro system in which to study the molecular mechanisms of KLF4 regulation and activity in macrophages. Our data indicate that KLF4 plays an important role is regulating immune responses to Mtb with the potential to both diminish host defense mechanisms and repress host-damaging immune mechanisms.
Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persi... more Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in...
CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) in dendritic ce... more CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) in dendritic cells. CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFNαβ. Because IFNαβ may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFNαβ. In our studies, CpG-B ODN inhibited induction of IFNαβ by CpG-A ODN, while induction of TNFα and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFNαβ was observed in Flt3L-induced murine DCs, purified murine mDCs and pDCs, and human PBMCs. CpG-B ODN inhibited induction of IFNαβ by agonists of multiple receptors, including MyD88-dependent TLRs and MyD88-independent receptors. CpG-B ODN did not inhibit the IFNαβ positive feedback loop “second wave” IFNαβ, since IFNαβ-induced expression of IFNαβ mRNA was unaffected, and CpG-B inhibition of IFNαβ was manifested in IFNαβR-/- DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early “first-wave” IFNα4 and IFNβ. Chrom...
Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety o... more Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety of immune responses. Pathogen-induced cell signaling and associated outcome research is abundant in macrophages, but minimal in dendritic cells. We investigated the role of ERK signaling in the induction of cytokines such as IL-12p40 and IL-10 following activation by Pam3Cys or Mycobacterium tuberculosis (Mtb) H37Ra. ERK ablation was accomplished pharmacologically with MERK1/2 inhibitor U0126 or genetically by using cells from Tpl2−/− mice (TPL2 connects TLR to ERK). We utilized western blotting to examine ERK activation, ELISA to assess cytokine production, and qRT-PCR to investigate mRNA levels. In macrophages, blockade of ERK signaling inhibited IL-10 production and increased IL-12p40 production (6–10 fold increase in IL-12p40 mRNA and 3–4 fold increase in protein). In dendritic cells, ERK blockade similarly inhibited IL-10 production, but produced a very different change in IL-12p40 e...
This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macropha... more This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macrophages. TLR2 signaling was induced with Pam3Cys, and the role of ERK signaling was interrogated pharmacologically with a MEK1/2 inhibitor (U0126) or genetically using bone-marrow-derived macrophages or dendritic cells from Tpl2−/− mice. We assessed cytokine production via ELISA and mRNA levels by qRT-PCR. In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited IL-10 production and increased IL-12p40 production significantly. In dendritic cells, blockade of ERK signaling similarly inhibited IL-10 production but decreased IL-12p40 production, opposite to the effect of ERK signaling blockade in macrophages. This difference in IL-12p40 regulation correlated with differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and dend...
The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we sh... more The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administrat...
TLR9 signaling induces type I IFN (IFN-I), which enhances MHC-I Ag cross processing, but TLR2 sig... more TLR9 signaling induces type I IFN (IFN-I), which enhances MHC-I Ag cross processing, but TLR2 signaling does not. Moreover, we previously reported that TLR2 signaling by Mycobacterium tuberculosis (Mtb) or other TLR2 agonists inhibited TLR9 induction of IFN-I and IFN-I-dependent MHC-I Ag cross processing (Simmons, 2010, J. Immunol.). In the current studies, a TLR2 agonist inhibited IFN-I mRNA induction when added together with TLR9 agonist for 2 to 24 h. TLR2 agonist inhibited TLR9-induced first wave IFN-I, since the inhibition was manifested in IFN-I receptor knock out DCs, which lack the second wave positive feedback mechanism, whereas second wave IFN-I mRNA induction by exogenous IFN-I was not inhibited. TLR2 also inhibited IFN-I induced by TLR7, another MyD88-dependent IFN-inducing receptor, but did not inhibit IFN-I induction by TLR3 or TLR4 (both TRIF-dependent, MyD88-independent). The inhibitory effect of TLR2 was not dependent on new protein synthesis or intercellular signaling. Current studies are further addressing the intracellular mechanism whereby TLR2 inhibits induction of IFN-I. This novel mechanism, whereby TLR2 inhbits IFN-I induction by TLR7/9, may shape immune responses to microbes that express ligands for both TLR2 and TLR7/TLR9, or responses to bacteria/virus coinfection.
Objectives To report methods and findings of 2 autopsies with molecular evaluation of severe acut... more Objectives To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals. Methods Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks. Results Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation. Conclusions SARS-CoV-2 testing and viral sequencin...
Krüppel-like factor 4 (KLF4) is a transcription factor that polarizes macrophages towards an anti... more Krüppel-like factor 4 (KLF4) is a transcription factor that polarizes macrophages towards an anti-inflammatory (M2) phenotype, which suggests that it may regulate immune responses to Mycobacterium tuberculosis (Mtb), as immune evasion by Mtb may be due to an overabundance of anti-inflammatory mediators in response to infection. We hypothesize that myeloid KLF4 is permissive of immune evasion by Mtb, causing decreased control of Mtb infection. There are no prior in vivo data on the role of KLF4 in infection. Using mice with myeloid-specific knockout of KLF4 (LysMCre/CreKLF4fl/fl, abbreviated Mye-KO) we observed decreased Mtb CFU in macrophage cultures in vitro and in lungs of Mye-KO mice early in infection (day 14) relative to wild-type controls. However, these experiments also revealed complexities to the KLF4-Mtb relationship. Despite their improved control of early infection, at later time points Mye-KO mice developed worsened clinical disease features, such as wasting, which suggests that the loss of KLF4 results in pathologic immune state. In addition, we found discrepancy between KLF4 RNA and protein expression during Mtb infection, suggesting that KLF4 is regulated at a posttranslational level during infection. We are designing an in vitro system in which to study the molecular mechanisms of KLF4 regulation and activity in macrophages. Our data indicate that KLF4 plays an important role is regulating immune responses to Mtb with the potential to both diminish host defense mechanisms and repress host-damaging immune mechanisms.
Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persi... more Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in...
CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) in dendritic ce... more CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) in dendritic cells. CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFNαβ. Because IFNαβ may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFNαβ. In our studies, CpG-B ODN inhibited induction of IFNαβ by CpG-A ODN, while induction of TNFα and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFNαβ was observed in Flt3L-induced murine DCs, purified murine mDCs and pDCs, and human PBMCs. CpG-B ODN inhibited induction of IFNαβ by agonists of multiple receptors, including MyD88-dependent TLRs and MyD88-independent receptors. CpG-B ODN did not inhibit the IFNαβ positive feedback loop “second wave” IFNαβ, since IFNαβ-induced expression of IFNαβ mRNA was unaffected, and CpG-B inhibition of IFNαβ was manifested in IFNαβR-/- DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early “first-wave” IFNα4 and IFNβ. Chrom...
Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety o... more Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety of immune responses. Pathogen-induced cell signaling and associated outcome research is abundant in macrophages, but minimal in dendritic cells. We investigated the role of ERK signaling in the induction of cytokines such as IL-12p40 and IL-10 following activation by Pam3Cys or Mycobacterium tuberculosis (Mtb) H37Ra. ERK ablation was accomplished pharmacologically with MERK1/2 inhibitor U0126 or genetically by using cells from Tpl2−/− mice (TPL2 connects TLR to ERK). We utilized western blotting to examine ERK activation, ELISA to assess cytokine production, and qRT-PCR to investigate mRNA levels. In macrophages, blockade of ERK signaling inhibited IL-10 production and increased IL-12p40 production (6–10 fold increase in IL-12p40 mRNA and 3–4 fold increase in protein). In dendritic cells, ERK blockade similarly inhibited IL-10 production, but produced a very different change in IL-12p40 e...
This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macropha... more This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macrophages. TLR2 signaling was induced with Pam3Cys, and the role of ERK signaling was interrogated pharmacologically with a MEK1/2 inhibitor (U0126) or genetically using bone-marrow-derived macrophages or dendritic cells from Tpl2−/− mice. We assessed cytokine production via ELISA and mRNA levels by qRT-PCR. In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited IL-10 production and increased IL-12p40 production significantly. In dendritic cells, blockade of ERK signaling similarly inhibited IL-10 production but decreased IL-12p40 production, opposite to the effect of ERK signaling blockade in macrophages. This difference in IL-12p40 regulation correlated with differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and dend...
The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we sh... more The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administrat...
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