MyD88 KO Mice Research Articles

IMMU-63. MESSENGER RNA CHALLENGE PREDICTS CANCER IMMUNOGENICITY AND RESPONSE TO CHECKPOINT INHIBITORS

Abstract BACKGROUND Checkpoint inhibition has emerged as a promising cancer therapeutic approach. Checkpoint inhibitors (CIs) function by overcoming the adaptive T cell exhaustion induced by solid tumor microenvironment including brain tumors, particularly in the neoadjuvant setting. Unfortunately, not every patient who receives CIs will unlock effective antitumor immunity. Objectives: Microsatellite instability and high mutational burden are considered key criteria to predict response to CIs. However, only a minority of patients will fulfill these criteria suggesting the need to develop innovative technologies to identify CI responsive patients. METHODS To determine whether glioma tumors will respond to CIs, we have developed a novel multilamellar mRNA-lipid particle tool to predict response ex vivo. Messenger RNA is complexed into multilamellar lipid particles to form aggregates (RNA-LPAs) to induce pathogen recognition receptor signaling in tumor cells to define an inflammatory profile as an indirect predictor to CI response. RESULTS In naïve mice, we found that multilamellar RNA-LPAs induce activation of Type I IFN system as supported by decreased secretion of IFN-α in MyD88 KO mice compared with wild type mice. We next selected murine glioma cell lines with known response profile to CIs (responders – GL261 and SMA560 vs non-responders – KR158b and CT2A). Prior to evaluation of cytokine profiling, successful uptake of RNA-LPAs was achieved in all cell lines. In 2D cell lines cultures and their corresponding 3D tumor spheroids, we demonstrated that treatment of CI responsive murine glioma models showed a differential cytokine signature (increased secretion of IFN-β/IL-6/CCL4 versus non-responsive models). CONCLUSION We identified a unique inflammatory signature after ex vivo RNA-LPAs challenge that correlates with checkpoint blockade activity. These findings allow for creation of a diagnostic assay to quickly assess tumor immunogenicity, allowing for informed immunotherapeutic treatment.

Abstract B048: Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4+ T cells-Myeloid cells axis

Abstract Introduction: Pancreatic cancer is a major cause of cancer-related mortality with a dismally low overall survival. Despite advancement in the cancer immunotherapy, pancreatic cancer remains unresponsive due to its immunosuppressives. We have established that lipopolysaccharide (LPS) reduces tumor burden in pancreatic cancer mouse-models by stimulating anti-tumor immunity and infiltrating cytotoxic CD4+ T-cells in the tumor-microenvironment. Here, we are investigating how LPS immuno-modulates pancreatic cancer niche and brings anti-cancerous effect. Methods: Pancreatic cancer-cells (KPCs) were implanted in C57BL/6, TRIF KO, MyD88 KO and TLR4 flox/flox LysmCre (TLR4f/f) mice subcutaneously and were treated with LPS (1mg/kg) weekly. The animals were sacrificed at endpoints and tumors were evaluated. Subcutaneous KPC-LPS model in BL/6 mice were also coupled with clodronate treatment to deplete cells of myeloid lineage. Tumor samples were analyzed using flow-cytometry and RT- PCR for immunocyte infiltration and expression of immune-associated genes. Dendritic cells (DCs) were isolated from BL/6 and TLR4f/f mice, pulsed with KPC cancer cell antigen, treated with LPS and taken to flow cytometry. Such DCs were also co-cultured with CD4+ T-cells, which were then subjected to cytotoxicity against cancer cells Results: LPS therapy in wild-type pancreatic cancer mouse-model significantly reduced the tumor burden by stimulating anti- tumor immunity in tumor-microenvironment and caused significant upregulation of activated DCs and macrophages. The tumor regressing effect of LPS disappeared in absence of TRIF as well as MyD88 receptors in TRIF KO and MyD88 KO mice respectively. Macrophage depletion by clodronate attenuated influence of LPS on tumor burden, which again proved futile in TLR4f/f model. On antigenic stimulation and LPS treatment, DCs from BL/6 mice expressed higher frequency of co-stimulatory molecules such as CD40, CD80 and CD86 whereas TLR4f/f mice derived DCs did not show such a trend. When such BL/6 DCs were co-cultured with CD4+ T- cells, they expressed higher cytotoxicity against cancer cells. Conclusions: LPS treatment reduces tumor burden in pancreatic cancer model by stimulating anti-tumor immunity in tumor- microenvironment wherein cells of the myeloid lineage are indispensable. This anti-cancer effect executed by CD4+ T-cells in an MHC II dependent manner, seems to be mediated through dendritic cells. Designer TLR4-agonists have the potential to emerges as a novel immunotherapeutic agent and could be combined with available immune-checkpoint blockade therapies. Citation Format: Utpreksha Vaish, Tejeshwar Jain, Dhanisha S Suresh, Maria F Salcedo-Noriega, Srikanth Iyer, Troy Randall, Vikas Dudeja. Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4T cells-Myeloid cells axis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B048.

MyD88 Signaling Accompanied by Microbiota Changes Supports Urinary Bladder Carcinogenesis.

Urinary bladder cancer (BC) inflicts a significant impairment of life quality and poses a high mortality risk. Schistosoma haematobium infection can cause BC, and the urinary microbiota of BC patients differs from healthy controls. Importantly, intravesical instillation of the bacterium Bacillus Calmette-Guerin stands as the foremost therapy for non-muscle invasive BC. Hence, studying the receptors and signaling molecules orchestrating bacterial recognition and the cellular response in the context of BC is of paramount importance. Thus, we challenged Toll-like receptor 4 (Tlr4) and myeloid differentiation factor 88 (Myd88) knock-out (KO) mice with N-butyl-N-(4-hydroxylbutyl)-nitrosamine (BBN), a well-known urinary bladder carcinogen. Gut microbiota, gene expression, and urinary bladder pathology were followed. Acute exposure to BBN did not reveal a difference in bladder pathology despite differences in the animal's ability to recognize and react to bacteria. However, chronic treatment resulted in reduced cancer invasiveness among Myd88KO mice while the absence of functional Tlr4 did not influence BC development or progression. These differences correlate with a heightened abundance of the Faecalibaculum genus and the lowest microbial diversity observed among Myd88KO mice. The presented data underscore the important role of microbiota composition and MyD88-mediated signaling during bladder carcinogenesis.

Maternal high fat diet exposure results in MyD88 dependent alteration in placental and offspring intestinal fatty acid transporters.

Abstract Introduction: We know that offspring born to parents with obesity are more likely to be obese. We hypothesized that the maternal high fat diet (mHFD) exposure may increase placental and offspring small intestinal transport of fatty acids. We hypothesized both these processes would be dependent on the presence of MyD88. Methods: Wild-type (WT) and MyD88 deficient mice on either 60% mHFD or regular diet (RD) were profiled. The baseline expression of fatty acid transport proteins FATP1 and FATP4, fatty acid binding proteins FABP1, FABP4, and CD36 were profiled by qRT-PCR from placenta after C-section at E20, and offspring small intestine 2-weeks after birth. Serum from 0-day old RD and HFD WT mice were collected and analyzed using high-throughput, non-targeted mass spectroscopy based metabolic analysis. Results: In WT mice, we found FATP1 was increased 32-fold and FABP was increased 7-fold in the mHFD placenta. FATP2 was decreased 2-fold and FATP4 was decreased 2.5-fold in the mHFD offspring small intestine. In MyD88 KO mice, we found no difference in any of the above fatty acid transporters. Serum of neonatal pups showed unique clustering of metabolites by PCA analysis in HFD and RD mice, with increased butanoate and propanoate metabolites in mHFD offspring. Conclusion: Fatty acid transport regulators in the placenta and offspring small intestine were modified by exposure mHFD. In both the placenta and small intestine, this outcome was dependent on the presence of MyD88. Fatty acid metabolites were increased in the serum of neonatal pups exposed to mHFD. These findings suggest that mHFD exposure can modulate fetal metabolism and may be an opportunity for future development of therapeutics that target placental and intestinal transporters.

Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds.

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO®, an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 μm in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 μm PTS remain unclear. Here, we use HALO® to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 μm PTS compared to 100 μm PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 μm PTS. When subcutaneously implanted in MyD88KO mice, 40 μm PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 μm PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.

The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model.

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 108 viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.

Insomnia and depressive behavior of MyD88-deficient mice: Relationships with altered microglial functions

Myeloid differentiation primary response gene 88 (MyD88) is essential for microglial activation. Despite the significant role of microglia in regulating sleep homeostasis, the contribution of MyD88 to sleep is yet to be determined. To address this, we performed electroencephalographic and electromyographic recordings on MyD88-KO mice and wild-type mice to investigate their sleep/wake cycles. In the daytime, MyD88-KO mice exhibited prolonged wakefulness and shorter non-rapid eye movement sleep duration. Tail suspension and sucrose preference tests revealed that MyD88-KO mice displayed a depressive-like phenotype. We determined monoamines in the prefrontal cortex (PFC) using high-performance liquid chromatography and observed a decreased content of serotonin in the PFC of MyD88-KO mice. Flow cytometry revealed that CD11b, CD45, and F4/80 expressions were elevated at Zeitgeber time (ZT) 1 compared to at ZT13 only in wild-type mice. Furthermore, MFG-E8 and C1qB-tagged synapses were enhanced at ZT1 in the PFC of wild-type mice but not in MyD88-KO mice. Primary cultured microglia from MyD88-KO mice revealed decreased phagocytic ability. These findings indicate that genetic deletion of MyD88 induces insomnia and depressive behavior, at least in part, by affecting microglial homeostasis functions and lowering the serotonergic neuronal output.

Host-to-Host Group A Streptococcus Transmission Causes Infection of the Lamina Propria but Not Epithelium of the Upper Respiratory Tract in MyD88-Deficient Mice.

To understand protective immune responses against the onset of group A Streptococcus respiratory infection, we investigated whether MyD88 KO mice were susceptible to acute infection through transmission. After commingling with mice that had intranasal group A Streptococcus (GAS) inoculation, MyD88-/- recipient mice had increased GAS loads in the nasal cavity and throat that reached a mean throat colonization of 6.3 × 106 CFU/swab and mean GAS load of 5.2 × 108 CFU in the nasal cavity on day 7. Beyond day 7, MyD88-/- recipient mice became moribund, with mean 1.6 × 107 CFU/swab and 2.5 × 109 CFU GAS in the throat and nasal cavity, respectively. Systemic GAS infection occurred a couple of days after the upper respiratory infection. GAS infects the lip, the gingival sulcus of the incisor teeth, and the lamina propria of the turbinate but not the nasal cavity and nasopharyngeal tract epithelia, and C57BL/6J recipient mice had no or low levels of GAS in the nasal cavity and throat. Direct nasal GAS inoculation of MyD88-/- mice caused GAS infection, mainly in the lamina propria of the turbinate. In contrast, C57BL/6J mice with GAS inoculation had GAS bacteria in the nasal cavity but not in the lamina propria of the turbinates. Thus, MyD88-/- mice are highly susceptible to acute and lethal GAS infection through transmission, and MyD88 signaling is critical for protection of the respiratory tract lamina propria but not nasal and nasopharyngeal epithelia against GAS infection.

Maternal high fat diet interacts with the maternal microbiome to expand IL-17 producing type 3 innate lymphoid cells in offspring.

Abstract Introduction: Previously, we demonstrated that maternal high fat diet (mHFD) exposure results in a unique pattern of microbial colonization in offspring and subsequent expansion of IL-17 producing type 3 innate lymphoid cells (ILC3). We hypothesized that in utero mechanisms involving the maternal microbiome were responsible for the phenotype in offspring. Methods: To assess if the maternal microbiome was responsible for altering the mHFD offspring phenotype, we delivered mHFD and regular diet (RD)-exposed pups at birth by C-section (CS) and exposed mHFD mothers to broad-spectrum antibiotics (ABX) throughout pregnancy. We cross-fostered the offspring to RD mothers at birth. Microbial colonization in 2-week-old offspring was determined by 16s-based rRNA analysis, intestinal cytokines were quantified by qRT-PCR and lamina propria cells (LP) were profiled by flow cytometry. To determine if innate immunity is altered prenatally by maternal diet, we delivered mHFD and RD offspring by CS and examined the small intestine (SI) at birth for cytokine expression, TLR signaling and LP profiling. Global MyD88 KO mice on mHFD or RD were also examined for the same endpoints. Results: mHFD offspring at 2-weeks showed an expansion of Firmicutes and IL-17 producing ILC3, independent of the mode of delivery. ABX in mHFD mothers ameliorated the mHFD offspring phenotype, with microbial colonization and ILC3 similar to controls. mHFD offspring had altered TLR signaling at birth in SI. ILC3 were expanded at birth in mHFD offspring (14.2% vs 9.5%). MyD88−/− offspring exposed to mHFD did not have an expansion of ILC3. Conclusion: mHFD prenatally alters the offspring microbiome and ILC3 and this effect is dependent on the maternal microbiome and TLR signaling.

Interleukin‐33 Dependent Endothelial Cell Hyperproliferation and Vascular Remodeling in Sugen/Hypoxia Mice

Pulmonary arterial hypertension (PAH) is a devastating disease that is often misdiagnosed and has a high mortality rate. It is characterized by remodeling of the small resistance arteries in the lungs that leads to elevated right ventricular pressure (RVP) and cardiac failure. Interleukin‐33 (IL‐33) is a cytokine that functions as an alarmin to sense danger or damage to endothelial cells, signal proliferation and restore barrier integrity. However, uncontrolled, active forms of IL‐33 could initiate endothelial hyperproliferation, a putative first step in the remodeling process. We hypothesize that IL‐33 initiates the hyperproliferative response of pulmonary artery endothelial cells and contributes to the wall remodeling and increased RVP observed in PAH. To test this, we induced pulmonary hypertension of C57Bl/6J (wild‐type), IL‐33 receptor knock‐out (ST2KO) and the adaptor protein, MyD88 knock‐out (MyD88KO) mice by exposure to 10% O2 and weekly SU5416 injections (SuHx). Hypertension was assessed by measuring RVP and arterial wall thickness (ratio of wall cross‐sectional area/total vessel cross‐sectional area). Proliferating pulmonary endothelial (CD31+/BrdU+) cells were detected by flow cytometry. Processed IL‐33 forms in the lung were analyzed by western blot. RVPs increased in wild‐type mice in response to SuHx (C57Bl/6J male, RA/DMSO 27.0 ± 3.0 mmHg, SuHx 40.5 ± 6.3 mmHg, p < 0.01; female, RA/DMSO 25.8 ± 3.3 mmHg, SuHx 43.8 ± 7.4 mmHg, p < 0.01) and, this response was attenuated in ST2KO mice (ST2KO male, RA/DMSO 25.0 ± 4.3 mmHg, SuHx 32.8 ± 6.9 mmHg, p<0.01; female, RA/DMSO 25.3 ± 3.5 mmHg, SuHx 32.8 ± 4.9 mmHg, p<0.01) and completely blocked in MyD88KO mice (MyD88KO male, RA/DMSO 19 ± 2.9 mmHg, SuHx 19.7 ± 5.9 mmHg, NS; female, RA/DMSO 20.2 ± 2.5 mmHg, SuHx 24.5 ± 7.1 mmHg, NS). Similarly, wall thickness increased in wild‐type mice, but not ST2KO and MyD88KO mice, under SuHx conditions (C57Bl/6J Male, RA/DMSO 0.35 ± 0.07, SuHx 0.53 ± 0.12, p < 0.01; Female RA/DMSO 0.34 ± 0.05, SuHx 0.41 ± 0.1, p=0.4; ST2KO Male, RA/DMSO 0.37 ± 0.07, SuHx 0.38 ± 0.13, NS; Female RA/DMSO 0.31 ± 0.02, SuHx 0.34 ± 0.03, NS; MyD88KO Male, RA/DMSO 0.37 ± 0.05, SuHx 0.41 ± 0.10, NS; Female RA/DMSO 0.34 ± 0.07, SuHx 0.38 ± 0.03, NS). The percentage of proliferating endothelial cells was increased in wild‐type mice under SuHx conditions (C57Bl/6J Male, RA/DMSO 1.9 ± 0.4, SuHx 4.8 ± 2.6, p< 0.01; Female RA/DMSO 0.3 ± 0.05, SuHx 0.4 ± 0.1, p= 0.07). This endothelial proliferative response was absent in ST2KO and Myd88KO mice (ST2KO Male, RA/DMSO 0.35 ± 0.07, SuHx 0.53 ± 0.12, NS; Female RA/DMSO 0.34 ± 0.05, SuHx 0.41 ± 0.1, NS). Proliferating (Ki‐67+) cells were detected in the intima layer of small arteries. The C‐terminal and caspase cleaved form of IL‐33 is increased in wild‐type and ST2KO mice but not in MyD88KO mice under SuHx conditions. IL‐33 signaling through the ST2 receptor is important for endothelial cell proliferation in the SuHx model. This response is associated with increased levels of both the C‐terminal IL‐1 like cytokine domain and caspase cleaved fragment of IL‐33 in lung tissue.Support or Funding InformationDepartment of Veteran Affairs

Inflammageing of Hematopoietic Stem Cells Is Driven By IL-1

Introduction: Lifelong blood production is sustained through a stepwise differentiation program by a limited number of self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). Hematopoietic cell development is tightly controlled by both cell-intrinsic and -extrinsic factors and its dysregulation can lead to aplasia or neoplasia. During ageing, HSCs increase in number, reduce self-renewal capacity on a per cell basis, skew towards myeloid differentiation, and show less efficient bone marrow (BM)-homing ability. We here evaluated how and to what extend HSC-extrinsic factors determine HSC behaviour during aging. Methods, Results and Discussion: To screen for aging-associated extrinsic factors, we performed antibody based protein arrays and transcriptome analysis with total BM of young (6-8 week old) versus aged (2 year old) animals. This demonstrated that RANTES, MIP-2, IL-1α and IL-1β are significantly upregulated in aged BM at both the protein as well as the RNA level. ELISA of peripheral blood (PB) serum and BM lysates indicated that IL-1α and IL-1β are locally increased and produced in BM, but are not significantly increased in PB serum. Further, qPCR of various BM cell types of hematopoietic (myeloid, lymphoid and progenitor cells) and non-hematopoietic/stromal origin indicated that multiple cell types upregulate Il1α and Il1β, with highest increase being derived from myeloid hematopoietic cells. This raised the possibility that elevated IL-1 is a result of an inflammatory response to circulating pathogen-derived compounds, possibly of bacterial origin. Indeed, we previously demonstrated that steady-state levels of granulopoiesis in young steady-state mice depend on heat-resistant microbiota-derived compounds (M.Balmer et al., The Journal of Immunology 2014). To prospectively test the role of IL-1-induced signalling and the microbiome during aging, we investigated the ageing-associated phenotype of HSCs in young and aged IL1RIKO mice and in young and aged germ-free mice. Both IL1RIKO and germ-free aged mice had lower counts of platelets and neutrophils in PB, and lower frequency of LT-HSCs (LKS Flt3-CD34-CD48-CD150+) in BM, compared to aged WT mice. Moreover, aged IL1RIKO LT-HSCs showed improved lymphoid lineage repopulation upon transplantation into lethally irradiated WT mice, compared to LT-HSCs of aged WT mice that demonstrated the known myeloid-biased lineage output. Interestingly, LT-HSCs from aged germ-free mice also demonstrated lymphoid-biased lineage differentiation as observed from young mice. In line with this finding, no difference was observed in IL-1α and IL-1β protein concentrations in BM lysates from young and aged germ-free mice. To test if IL-1 increase in aged steady-state mouse BM is indeed dependent on ligation of pattern recognition receptors and consecutive signalling, we analysed MyD88 and Trif KO mice, respectively. Both aged KO mice showed compared to WT mice reduced BM IL-1 levels and a reduced ageing-phenotype of HSCs, with the most profound difference in Trif KO mice. Interestingly, this correlates with our previous finding on pathogen-Induced TLR4-TRIF innate immune signaling in HSCs, inducing reduced competitive fitness (Takizawa et al., Cell Stem Cell 2017). Conclusions: Our data demonstrate that ageing associated phenotype and myeloid-biased differentiation of HSCs is a result of signals derived from the microbiome, that act through increased IL-1 signalling, locally in BM. Disclosures No relevant conflicts of interest to declare.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by Bcell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including Tcells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that Bcell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Excision release of 5?hydroxycytosine oxidatively induced DNA base lesions from the lung genome by cat dander extract challenge stimulates allergic airway inflammation.

Ragweed pollen extract (RWPE) induces TLR4-NFκB-CXCL-dependent recruitment of ROS-generating neutrophils to the airway and OGG1 DNA glycosylase-dependent excision of oxidatively induced 8-OH-Gua DNA base lesions from the airway epithelial cell genome. Administration of free 8-OH-Gua base stimulates RWPE-induced allergic lung inflammation. These studies suggest that stimulation of innate receptors and their adaptor by allergenic extracts initiates excision of a set of DNA base lesions that facilitate innate/allergic lung inflammation. To test the hypothesis that stimulation of a conserved innate receptor/adaptor pathway by allergenic extracts induces excision of a set of pro-inflammatory oxidatively induced DNA base lesions from the lung genome that stimulate allergic airway inflammation. Wild-type (WT), Tlr4KO, Tlr2KO, Myd88KO, and TrifKO mice were intranasally challenged once or repeatedly with cat dander extract (CDE), and innate or allergic inflammation and gene expression were quantified. We utilized GC-MS/MS to quantify a set of oxidatively induced DNA base lesions after challenge of naïve mice with CDE. A single CDE challenge stimulated innate neutrophil recruitment that was partially dependent on TLR4 and TLR2, and completely on Myd88, but not TRIF. A single CDE challenge stimulated MyD88-dependent excision of DNA base lesions 5-OH-Cyt, FapyAde, and FapyGua from the lung genome. A single challenge of naïve WT mice with 5-OH-Cyt stimulated neutrophilic lung inflammation. Multiple CDE instillations stimulated MyD88-dependent allergic airway inflammation. Multiple administrations of 5-OH-Cyt with CDE stimulated allergic sensitization and allergic airway inflammation. We show for the first time that CDE challenge stimulates MyD88-dependent excision of DNA base lesions. Our data suggest that the resultant-free base(s) contribute to CDE-induced innate/allergic lung inflammation. We suggest that blocking the MyD88 pathway in the airways with specific inhibitors may be a novel targeted strategy of inhibiting amplification of innate and adaptive immune inflammation in allergic diseases by oxidatively induced DNA base lesions.

MyD88 controls airway epithelial Muc5ac expression during TLR activation conditions from agricultural organic dust exposure.

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

Elucidating synergistic and antagonistic effects of B cell receptor and toll-like receptors 3, 4, and 9 in B cell activation

Abstract B cells are key components of the humoral immune response. Two forms of B cell activation come from engagement of the B cell receptor (BCR) with foreign antigens and from pathogen-associated molecular patters (PAMPs) binding to toll-like receptors (TLRs). For TLRs, MyD88 and TRIF are the major adaptors that lead to NFkB and IRF3 activation respectively. Among various TLRs that are present in B cells, TLR9 is only able to use MyD88 and TLR3 only signals through TRIF, while TLR4 uses both pathways. Therefore, these TLRs provide excellent tools to investigate the roles of TRIF and MyD88 pathways in B cell function and the interplay between them. Here, using stimulation through these three different TLRs either individually or in combination with each other and with/or without anti-IgM, we observed that there is synergy and antagonism between these pathways in terms of activation induced metabolic boost, cytokine production, IgM secretion, expression of various cell surface markers, and antigen presentation. We identified the TRIF pathway as an inducer of Type-I interferon secretion by B cells, which eventually favors the expression of costimulatory molecules such as CD86. On the other hand, MyD88 stimulation antagonized the expression of costimulatory molecules and dramatically induced rapid proliferation and production of IgM as well as proinflammatory cytokines such as IL-6 and TNF. Using B cells obtained from MyD88 or TRIF KO mice, we showed that the interplay between pathways is of limited scale; however, for TLR4 signaling MyD88 has a largely permissive effect while TRIF determines the magnitude of response. These results further our understanding of the complex TLR signaling machinery in B lymphocytes.

Establishment of a mouse model of pneumonia with an isolated epidemic ST23 Klebsiella pneumonia strain

Objective To establish a mouse model of pneumonia with C57BL/6 and MyD88KO mice after infection with an isolated ST23 Klebsiella pneumonia (KP) strain, which was an epidemic strain and identified by multilocus sequence typing (MLST). Methods Fifty C57BL/6 mice were randomly divided into three groups: KP infection, control and immunosuppressive groups. Thirty MyD88KO mice were divided into KP infection and control groups. All mice in the KP infection groups were infected with 50 μl of ST23 KP strain through nasal dripping. Equal volume of PBS was used to set up the control groups. Mice in the immunosuppressive group were first injected with cyclophosphamide for three days and then infected with equal volume of ST23 KP strains through nasal dripping. Clinical signs and survival curves during KP infection were monitored. Moreover, pulmonary bacterial loads and histopathological changes in the KP-infected mice were detected at different time points. Results ST23 KP-infected C57BL/6 mice showed inflammatory cell infiltration in lung tissues on the 10th day and remained alive on the 21st day. All ST23 KP-infected MyD88KO mice died on the 5th day with severe histopathological damage in lung tissues. C57BL/6 mice that pretreated with cyclophosphamide had similar symptoms with MyD88KO mice after infection and died on the 5th day. Some critical inflammatory mediators such as TNF-a, nitric oxide synthase 1 (NOS1) and NF-κBp65 were up-regulated in lung tissues of mice after KP infection. No inflammatory syndromes were found in the mice of PBS control groups. Conclusion This study suggests that the mouse model of pneumonia is successfully established with KP strain. It will help researchers to study the characteristics and pathogenesis of ST23 KP strain-induced pneumonia and to seek safe treatments in the future. Key words: Klebsiella pneumonia (KP); ST23; TNF-α; NF-κBp65

MMP2 and MMP9 are Associated with Apical Periodontitis Progression and Might be Modulated by TLR2 and MyD88.

The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.

Sepsis Upregulates CD14 Expression in a MyD88-Dependent and Trif-Independent Pathway.

An overwhelming immune response, particularly from macrophages, plays a critical role in survival and organ damage in sepsis patients. Toll-like receptors (TLRs) are important receptors to recognize the conserved motifs expressed by invading bacteria. The TLRs except TLR3 signal via a MyD88-dependent pathway. TLR3 uses a TRIF-dependent pathway, while TLR4 uses both MyD88 and TRIF-dependent pathways. Previous studies indicated that CD14 was necessary for TLRs-dependent production of pro-inflammatory cytokines. Blocking CD14 protected against the deleterious systemic inflammatory response associated with sepsis. The aim of this study was to determine the signaling pathway of TLR activation-induced CD14 expression in models of polymicrobial sepsis and in peritoneal macrophages. We found that CD14 expression was upregulated in the lung, liver, and kidney of septic mice induced by cecal ligation puncture. In cultured peritoneal macrophages, specific agonists for all TLRs, except for TLR3, increased CD14 expression. Lipopolysaccharide-induced upregulation of CD14 was abolished in peritoneal macrophages from MyD88 KO mice but increased in TRIF inhibitor, resveratrol pretreated wild-type macrophages. Moreover, MyD88 KO, but not TRIF KO mice, showed a decreased CD14 expression in the tissue of septic mice, which was associated with a strongly attenuated inflammatory response and increased survival rate. These data suggest that a MyD88-dependent and TRIF-independent pathway of TLR is activated in upregulating CD14 expression under septic conditions. This study deciphers a critical cross-talk between TLRs and CD14.

Dectin-1 Activation Exacerbates Obesity and Insulin Resistance in the Absence of MyD88

SUMMARYThe underlying mechanism by which MyD88 regulates the development of obesity, metainflammation, and insulin resistance (IR) remains unknown. Global deletion of MyD88 in high-fat diet (HFD)-fed mice resulted in increased weight gain, impaired glucose homeostasis, elevated Dectin-1 expression in adipose tissue (AT), and proinflammatory CD11c+ AT macrophages (ATMs). Dectin-1 KO mice were protected from diet-induced obesity (DIO) and IR and had reduced CD11c+ AT macrophages. Dectin-1 antagonist improved glucose homeostasis and decreased CD11c+ AT macrophages in chow- and HFD-fed MyD88 KO mice. Dectin-1 agonist worsened glucose homeostasis in MyD88 KO mice. Dectin-1 expression is increased in AT from obese individuals. Together, our data indicate that Dectin-1 regulates AT inflammation by promoting CD11c+ AT macrophages in the absence of MyD88 and identify a role for Dectin-1 in chronic inflammatory states, such as obesity. This suggests that Dectin-1 may have therapeutic implications as a biomarker for metabolic dysregulation in humans.

MyD88 in donor bone marrow cells is critical for protection from acute intestinal graft-vs.-host disease.

To understand the role of myeloid differentiation factor 88 (MyD88) expressed by donor bone marrow (BM) in the pathophysiology of graft-vs.-host disease (GVHD), we investigated the effects of transplantation of MyD88-deficient T cell-depleted BM (MyD88KO TCD-BM) on the severity of GVHD. Transplantation with MyD88KO TCD-BM aggravated GVHD; serious gut damage was evident, with high infiltration of T cells into the intestines of recipients and markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs). MDSCs from MyD88KO mice were defective in inducing donor T-cell apoptosis and inhibiting T-cell proliferation. Supplementation of transplanted mice with MDSCs from wild-type mice, but not MyD88KO mice, attenuated GVHD severity with reduced intestinal T-cell infiltration in MyD88KO TCD-BM recipients. Pretreatment of BM donors with lipopolysaccharide to increase MDSC levels and MyD88 transcription in the TCD-BM transplant alleviated GVHD severity and intestinal T-cell infiltration. The T cell/MDSC ratios were correlated with intestinal GVHD severity in both animal models and human patients. This study indicates that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal intestinal GVHD.