Loss Of MyD88 Research Articles

Myeloid MyD88 restricts CD8+ T cell response to radiation therapy in pancreatic cancer

Radiation therapy induces immunogenic cell death in cancer cells, whereby released endogenous adjuvants are sensed by immune cells to direct adaptive immune responses. TLRs expressed on several immune subtypes recognize innate adjuvants to direct downstream inflammatory responses in part via the adapter protein MyD88. We generated Myd88 conditional knockout mice to interrogate its contribution to the immune response to radiation therapy in distinct immune populations in pancreatic cancer. Surprisingly, Myd88 deletion in Itgax (CD11c)-expressing dendritic cells had little discernable effects on response to RT in pancreatic cancer and elicited normal T cell responses using a prime/boost vaccination strategy. Myd88 deletion in Lck-expressing T cells resulted in similar or worsened responses to radiation therapy compared to wild-type mice and lacked antigen-specific CD8+ T cell responses from vaccination, similar to observations in Myd88−/− mice. Lyz2-specific loss of Myd88 in myeloid populations rendered tumors more susceptible to radiation therapy and elicited normal CD8+ T cell responses to vaccination. scRNAseq in Lyz2-Cre/Myd88fl/fl mice revealed gene signatures in macrophages and monocytes indicative of enhanced type I and II interferon responses, and improved responses to RT were dependent on CD8+ T cells and IFNAR1. Together, these data implicate MyD88 signaling in myeloid cells as a critical source of immunosuppression that hinders adaptive immune tumor control following radiation therapy.

Abstract 6410: Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors

Abstract The balance of innate signaling through adaptor proteins such as MyD88 and TRIF is critical in directing the pattern of inflammatory responses following exposure to endogenous adjuvants. While innate stimuli can cause inflammation that supports adaptive immunity, cancer myeloid cells can be pre-polarized such that the same inflammatory signals cause myeloid cells to suppress adaptive immune responses. We aim to investigate the signals regulating type I IFN secretion by innate immune cells in tumors in order to improve type I IFN secretion, activation of innate immune cells, direct macrophage polarization and improve CD8+ T cell mediated anti-tumor immunity. To better understand the role of MyD88-mediated signaling in driving response to innate adjuvants released following RT in solid tumors, we developed MyD88 conditional knockout mouse models. MyD88 was deleted specifically in CD11c expressing dendritic cells (DC), Lck expressing T cells, or Lyz2 expressing myeloid cells that include macrophages, monocytes and granulocytes. Mice bearing pancreatic tumors (Panc02-SIY or PK5L1940) were randomized to no treatment or 16 Gy CT-guided RT and followed for treatment responses. Lyz2-Cre/MyD88fl/fl mice demonstrated improved responses to RT in pancreatic tumors as compared to control MyD88fl/fl mice, or Lck-Cre mice. The improved responses in Lyz2-Cre/MyD88fl/fl mice were shown to be dependent on CD8+ T cells as well as on type I IFN signaling. To analyze mechanisms of response, tumors were digested into a single cell suspension and CD45+ cells were isolated for single cell sequencing three days post-RT. Lyz2-Cre/MyD88fl/fl mice showed diminished Th1 and Th2-type T cells and had a higher M1/TAM ratio compared to MyD88fl/fl mice. Loss of MyD88 in myeloid cells resulted in increased activation of IFN-dependent transcriptional responses in multiple immune cell populations in the tumor. To model responses ex vivo, bone marrow-derived macrophages (BMDMs) were activated with lipopolysaccharide (LPS) or a synthetic cyclic dinucleotide (CDA) for analysis of cytokine secretion in either monolayer or 3D spheroid culture conditions. BMDM derived from Lyz2-Cre/MyD88fl/fl cultured as a monolayer showed significantly altered secretion of TNF-α, IL-10 and IL-6 on activation with LPS as compared to controls. Preliminary data obtained from 3D spheroid culture systems demonstrated that 3D interactions between cancer cells and macrophages resulted in a significant increase in BMDM co-stimulatory phenotype following activation with innate stimuli compared to 2D cultures. These data suggest that conventional innate signaling through MyD88 in myeloid populations suppresses IFN production and adaptive immune control of irradiated tumors. 3D cultures are an effective tool to study cellular interactions ex vivo and screen novel interventions prior to in vivo translation. Citation Format: Aanchal Preet Kaur, Terry R. Medler, Tiffany C. Blair, Alejandro F. Alice, Megan R. Ball, Alexa K. Dowdell, Brian D. Piening, Marka R. Crittenden, Michael J. Gough. Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6410.

Abstract 655: Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells

Abstract Programmed cell death (PCD) was originally limited to apoptosis, but now includes pyroptosis, ferroptosis, necroptosis, and paraptosis. Necroptosis has attracted particular interest as an approach for immunotherapy as activation of the RIPK1/RIPK3 necrosome phosphorylates MLKL to form membrane pores, leading to loss of integrity and enhanced ability to induce an antigen-specific immune response in models of vaccination. Here we sought to determine if necroptosis within tumor cells was an important regulatory of anti-tumor immunity. Deficiency in Ripk3 or Mlkl did not impact tumor incidence or growth in genetic models of mammary carcinoma or lung adenocarcinoma. Similarly, the growth of syngeneic cell lines was not altered by loss of Ripk3 or Mlkl in vitro or in vivo. We next examined the induction of apoptosis or necroptosis in syngeneic tumor models using genetic constructs with doxycycline-controlled expression. In line with published data, subcutaneous vaccination demonstrated that RIPK3-induced necroptosis promoted an antigen-specific CD8+ T cell response greater than that observed with caspase 8-induced apoptosis. Surprisingly however, when necroptosis was induced within established PyMT-B6 orthotopic mammary tumors, we observed significantly reduced survival, which correlated with higher neutrophil and macrophage recruitment, along with lower CD8+ T cell infiltration and IFN-γ expression. In vitro, necroptosis induced with either RIPK3 or MLKL greatly enhanced expression of Cxcl1 and Cxcl2, a property that could be recapitulated by transferring dead cell supernatant to live cells, suggesting the presence of an alarmin or damage-associated molecular pattern (DAMP). We therefore knocked out MyD88, which serves as a critical adapter protein to a number of surface receptors, including the TLR family, the IL1 receptor, and RAGE. The loss of MyD88 completely eliminated the ability of tumor cells to express Cxcl1 in response to supernatant from necrotic cells. Expression was not driven by release of HMGB1 or signaling through RAGE or TLRs. Instead, we found that blocking the interleukin (IL)1 receptor or IL1α, but not IL1β, prevented Cxcl1 expression. IL1α is a constitutively expressed by some tumors, suggesting a potential role for IL1α in regulating immune responses to therapies that induce necrotic-like forms of cell death. In support of this, Il1a expression was strongly predictive of progression free survival in triple-negative breast cancer. These findings suggest that IL1α has an unexplored role in regulating the tumor microenvironment. By understanding how IL1α regulates the immune response during cytotoxic therapy, our findings may explain why certain therapies are immunogenic (or not) in different cancers. Citation Format: Kay Hänggi, Aysenur Keske, Alycia Gardner, Jie Li, Olabisi Osunmakinde, Daiana Celias, Amer Beg, Brian Ruffell. Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 655.

Loss of MyD88 in microglia impacts cognition and parvalbumin cell density in the male brain

Loss of MyD88 in microglia impacts cognition and parvalbumin cell density in the male brain

Maternal B cell signaling orchestrates fetal development in mice.

B cell participation in early embryo/fetal development and the underlying molecular pathways have not been explored. To understand whether maternal B cell absence or impaired signaling interferes with placental and fetal growth, we paired CD19-deficient (CD19-/-) mice, females with B cell-specific MyD88 (BMyD88-/-) or IL10 (BIL10-/-) deficiency as well as wild-type and MyD88-/- controls on C57Bl/6 background with BALB/c males. Pregnancies were followed by ultrasound and Doppler measurements. Implantation number was reduced in BMyD88-/- and MyD88-/- mice. Loss of MyD88 or B cell-specific deletion of MyD88 or IL10 resulted in decreased implantation areas at gestational day (gd) 5, gd8 and gd10, accompanied by reduced placental thickness, diameter and areas at gd10. Uterine artery resistance was enhanced in BIL10-/- dams at gd10. Challenge with 0.4 mg lipopolysaccharide/kg bodyweight at gd16 revealed that BMyD88-/-, BIL10-/- and CD19-/- mothers delivered preterm, whereas controls maintained their pregnancy. B cell-specific MyD88 and IL10 expression is essential for appropriate in utero development. IL10+B cells are involved in uterine blood flow regulation during pregnancy. Finally, B cell-specific CD19, MyD88 and IL10 expression influences susceptibility towards preterm birth.

Induction of IL-12p40 and type 1 immunity by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade.

Toxoplasma gondii is an orally acquired pathogen that induces strong IFN-γ based immunity conferring protection but that can also be the cause of immunopathology. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. Here we focus on induction of less well understood immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, in particular as they occur in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88-/- background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. Infection-induced IL-12 was lower in the absence of MyD88, but was still clearly above noninfected levels. Overall, this carried through to the IFN-γ response, which while generally decreased was still remarkably robust in the absence of MyD88. In the latter mice, IL-12 was strictly required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4+ T cells, and CD8+ T cells each contributed to the IFN-γ pool. We report that ILC3 were expanded in infected MyD88-/- mice relative to their MyD88+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. Furthermore, bacterial flagellin and Toxoplasma specific CD4+ T cell populations in the lamina propria expanded in response to infection in both WT and KO mice. Finally, we show that My88-independent IL-12 and T cell mediated IFN-γ production require the presence of the intestinal microbiota. Our results identify MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, our data reveal an underlying network of immune responses that do not involve signaling through MyD88.

IL-12p40 and Type 1 immunity are induced by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade

Abstract Toxoplasma gondii is an orally acquired pathogen that induces strong, protective IFN-γ based immunity. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. The objective of this study was to determine the induction of immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, with particular focus on events in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88−/− background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. While infection-induced IL-12 was lower in the absence of MyD88, the cytokine was nonetheless required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4+ T cells, and CD8+ T cells each contributed to the IFN-γ pool. Furthermore, bacterial flagellin and Toxoplasma specific CD4+T cell populations in the lamina propria expanded to similar frequencies observed in the wildtype small intestine. ILC3 were expanded in both naïve and infected MyD88−/− mice relative to their MyD88+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. This study identifies MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity, and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, these data reveal an underlying network of immune responses that do not involve MyD88 but that contribute to pathways associated with resistance to T. gondii.

4323 Inflammation partially mediates fatigue-like behavior in mice

OBJECTIVES/GOALS: Fatigue is a distressing side effect of cancer and its treatment. It is a subjective symptom that can include mental, physical, emotional, and motivational components. We sought to determine whether preventing inflammation affects fatigue-like behavior in a mouse model of radiation therapy. METHODS/STUDY POPULATION: C57BL/6 mice received three consecutive 8 gray doses of daily peripheral irradiation. We used voluntary wheel running activity to measure fatigue-like behavior before and after this period. Minocycline, an antibiotic with anti-inflammatory effects, was administered beginning a week before irradiation and continued throughout the experiment. We also tested mice lacking the toll-like receptor adaptor protein, MyD88. Cognitive abilities were tested using spontaneous alternation in a Y-maze. RESULTS/ANTICIPATED RESULTS: We found that minocycline reduces fatigue-like behavior exhibited after irradiation, but had no effect on pre-irradiation activity levels. Similarly, fatigue-like behavior after radiation was partially reversed by genetic loss of MyD88. Y-maze spontaneous alternation performance remained similar in all groups. DISCUSSION/SIGNIFICANCE OF IMPACT: Both pharmacological and genetic anti-inflammatory manipulations increased voluntary activity levels after irradiation. Our results suggest that inflammation is an important factor in the development of fatigue-like behavior. Modulators of inflammatory processes hold potential for alleviating fatigue.

Ikaros prevents autoimmunity by controlling anergy and Toll-like receptor signaling in B cells.

The establishment of a diverse B cell antigen receptor (BCR) repertoire by V(D)J recombination also generates autoreactive B cells. Anergy is one tolerance mechanism; it renders autoreactive B cells insensitive to stimulation by self-antigen, whereas Toll-like receptor (TLR) signaling can reactivate anergic B cells. Here, we describe a critical role of the transcription factor Ikaros in controlling BCR anergy and TLR signaling. Mice with specific deletion of Ikaros in mature B cells developed systemic autoimmunity. Ikaros regulated many anergy-associated genes, including Zfp318, which is implicated in the attenuation of BCR responsiveness by promoting immunoglobulin D expression in anergic B cells. TLR signaling was hyperactive in Ikaros-deficient B cells, which failed to upregulate feedback inhibitors of the MyD88-nuclear factor κB signaling pathway. Systemic inflammation was lost on expression of a non-self-reactive BCR or loss of MyD88 in Ikaros-deficient B cells. Thus, Ikaros acts as a guardian preventing autoimmunity by promoting BCR anergy and restraining TLR signaling.

MyD88 signaling in T regulatory cells by endogenous ligands dampens skin inflammation in filaggrin deficient mice

Mutations in filaggrin are associated with atopic dermatitis. Filaggrin-deficient flaky tail (Flgft/ft) mice develop spontaneous inflammatory skin lesion that wax and wane. We show that loss of MyD88 promotes the persistence of skin lesions in Flgft/ft mice and exaggerates their expression of the Th17-associated cytokines Il7a and Il22. The development and persistence of skin lesions in Flgft/ft mice was independent of the microbiota. MyD88-mediated signals are shown to be important for the accumulation of T regulatory cells (Tregs) in lesional skin of Flgft/ft mice. Adoptive transfer of WT Tregs dampened the severity of skin lesions in MyD88−/−/Flgft/ft mice. These results suggest that MyD88 signaling in Treg cells by endogenous ligands attenuates skin inflammation in filaggrin deficiency.

MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Epithelial Myeloid-Differentiation Factor 88 Is Dispensable during Klebsiella Pneumonia.

Klebsiella pneumoniae is a common cause of pneumonia. Previous studies have documented an important role for Toll-like receptors (TLRs) expressed by myeloid cells in the recognition of K. pneumoniae and the initiation of a protective immune response. Lung epithelial cells also express TLRs and can participate in innate immune defense. The aim of this study was to examine the role of the common TLR adaptor protein myeloid-differentiation factor (MyD) 88 in lung epithelium during host defense against K. pneumoniae-induced pneumonia. To this end, we first crossed mice expressing cre recombinase under the control of the surfactant protein C (SftpCcre) or the club cell 10 kD (CC10cre) promoter with reporter mice to show that SftpCcre mice mainly express cre in type II alveolar cells, whereas CC10cre mice express cre almost exclusively in bronchiolar epithelial cells. We then generated mice with cell-targeted deletion of MyD88 in type II alveolar (SftpCcre-MyD88-lox) and bronchiolar epithelial (CC10cre-MyD88-lox) cells, and infected them with K. pneumoniae via the airways. Bacterial growth and dissemination were not affected by the loss of MyD88 in SftpCcre-MyD88-lox or CC10cre-MyD88-lox mice compared with control littermates. Furthermore, inflammatory responses induced by K. pneumoniae in the lung were not dependent on MyD88 expression in type II alveolar or bronchiolar epithelial cells. These results indicate that MyD88 expression in two distinct lung epithelial cell types does not contribute to host defense during pneumonia caused by a common human gram-negative pathogen.

Uropathogenic Escherichia coli causes fibrotic remodelling of the epididymis.

Despite antibiotic treatment, up to 40% of patients have impaired fertility after epididymitis due to serovars of Escherichia coli, a frequent pathogen. The reasons for infertility are unclear, but it may result from epididymal duct obstruction. To determine whether E. coli infection of the epididymis causes obstruction due to fibrosis, and to identify the key mediators, tissues from patients with epididymitis were assessed. Additionally, epididymitis was induced with uropathogenic E. coli (UPEC) or commensal serovars in wild-type and MyD88(-/-) mice, which are relatively unresponsive to bacterial pathogens. Epididymal organ cultures were treated with activin A and bacteria and their histology and levels of cytokines and fibrosis markers were analysed. Patients with epididymitis showed severe fibrosis of the epididymal duct. In mice, UPEC infection also caused fibrosis and ductal obstruction in the cauda epididymis. Levels of mRNA for fibrotic markers (α-smooth muscle actin, fibronectin) and cytokines (activin A, TNFα, IL-1α, IL-1β, IL-6) and total collagen levels were significantly elevated. This fibrotic response was blunted by the loss of MyD88. Activin A induced fibrosis in cultured epididymis, which was inhibited by the activin-binding protein follistatin. In summary, bacterial epididymitis causes fibrosis and obstruction. The milder tissue damage in Myd88(-/-) UPEC epididymitis highlights the importance of the host response to infection in causing epididymal damage. Elevated levels of activin A in vivo and fibrotic remodelling elicited by activin A in vitro indicate that this cytokine is a potential target for supplementary treatment to antibiotic therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Loss of MyD88 alters neuroinflammatory response and attenuates early Purkinje cell loss in a spinocerebellar ataxia type 6 mouse model.

Spinocerebellar ataxia type 6 (SCA6) is dominantly inherited neurodegenerative disease, caused by an expansion of CAG repeat encoding a polyglutamine (PolyQ) tract in the Cav2.1 voltage-gated calcium channel. Its key pathological features include selective degeneration of the cerebellar Purkinje cells (PCs), a common target for PolyQ-induced toxicity in various SCAs. Mutant Cav2.1 confers toxicity primarily through a toxic gain-of-function mechanism; however, its molecular basis remains elusive. Here, we studied the cerebellar gene expression patterns of young Sca6-MPI118Q/118Q knockin (KI) mice, which expressed mutant Cav2.1 from an endogenous locus and recapitulated many phenotypic features of human SCA6. Transcriptional signatures in the MPI118Q/118Q mice were distinct from those in the Sca1154Q/2Q mice, a faithful SCA1 KI mouse model. Temporal expression profiles of the candidate genes revealed that the up-regulation of genes associated with microglial activation was initiated before PC degeneration and was augmented as the disease progressed. Histological analysis of the MPI118Q/118Q cerebellum showed the predominance of M1-like pro-inflammatory microglia and it was concomitant with elevated expression levels of tumor necrosis factor, interleukin-6, Toll-like receptor (TLR) 2 and 7. Genetic ablation of MyD88, a major adaptor protein conveying TLR signaling, altered expression patterns of M1/M2 microglial phenotypic markers in the MPI118Q/118Q cerebellum. More importantly, it ameliorated PC loss and partially rescued motor impairments in the early disease phase. These results suggest that early neuroinflammatory response may play an important role in the pathogenesis of SCA6 and its modulation could pave the way for slowing the disease progression during the early stage of the disease.

Deletion of FADD in macrophages and granulocytes results in RIP3- and MyD88-dependent systemic inflammation.

Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.

Loss of MyD88 leads to more aggressive TRAMP prostate cancer and influences tumor infiltrating lymphocytes.

The influence of pattern recognition receptor (PRR) signaling in the prostate tumor microenvironment remains unclear. Although there may be a role for PRR agonists as adjuvants to therapy, prior evidence suggests tumor promoting as well as tumor inhibiting mechanisms. The purpose of this study is to examine the role of the key Toll-like receptor (TLR) signaling adaptor protein myeloid differentiation primary response gene 88 (MyD88) in prostate cancer development. MyD88(-/-) mice in a C57Bl6 background were crossed with transgenic adenocarcinomas of the mouse prostate (TRAMP) mice to create MyD88(-/-) TRAMP(Tg+/-) animals, which were compared to MyD88(+/+) TRAMP(Tg+/-) animals and their non-transgenic counterparts at 30 weeks. Prostates were examined histologically, by immunohistochemistry and immunofluorescence staining, and by qPCR, to characterize tumor-infiltrating immune populations as well as activation of the downstream NF-κB pathway and androgen receptor (AR) expression. Splenocytes were examined for development of distinct immune cell populations. Absence of MyD88 led to increased prostatic intraepithelial neoplasm (PIN) and areas of well-differentiated adenocarcinoma in TRAMP transgenic mice. Analysis of infiltrating immune populations revealed an increase in CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), as evidenced by increased expression of prostatic arginase-1 and iNOS as well as the cytokine IL-10, and a deficiency in NK cells in prostates from MyD88(-/-) TRAMP(Tg+/-) compared to MyD88(+/+) TRAMP(Tg+/-) mice, whereas a decrease in splenocytic NK cell differentiation was observed in MyD88(-/-) mice. Prostate tumors revealed no significant differences in NF-κB or AR expression in MyD88(+/+) TRAMP(Tg+/-) compared to MyD88(-/-) TRAMP(Tg+/-) mice. During prostate cancer development in the TRAMP model, MyD88 may play a role in limiting prostate tumorigenesis by altering tumor-infiltrating immune populations. This suggests that in the context of specific cancers, distinct PRRs and signaling pathways of innate immune signaling may influence the tumor microenvironment and represent a novel therapeutic strategy.

A narrow repertoire of transcriptional modules responsive to pyogenic bacteria is impaired in patients carrying loss-of-function mutations in MYD88 or IRAK4.

Loss of function in the kinase IRAK-4 or the adapter MyD88 in humans interrupts a pathway critical for pathogen sensing and ignition of inflammation. Yet patients with loss of function mutations are surprisingly only susceptible to a limited range of pathogens. We employed a systems approach to investigate transcriptome responses following in vitro exposure of patients’ blood to Toll-like receptor and interleukin-1 receptor agonists, and whole pathogens. Responses to purified agonists were globally abolished but variable residual responses were present following exposure to whole pathogens. Further dissection of the latter responses identified a narrow repertoire of immune transcriptional programs affected by loss of MyD88 or IRAK-4 function. This work introduces the use of a systems approach for the global assessment of innate immune responses, and the characterization of human primary immunodeficiencies.

MyD88-mediated TLR signaling protects against acute rotavirus infection while inflammasome cytokines direct Ab response.

Rotavirus (RV) infects small intestinal epithelial cells, inducing severe diarrhea in children, resulting in over 500,000 deaths annually. Relatively little is known about how innate immunity contains acute infection and drives adaptive immune responses that afford complete clearance of RV and protection against future infection. Hence, we examined the consequence of the absence of MyD88, known to be central to innate immunity, in a mouse model of RV infection. The absence of MyD88, but not combined blockade of IL-1β and IL-18 signaling, resulted in greater infectivity, as reflected by levels of RV in feces, intestinal lysates and viremia. Such increased RV levels correlated with an increase in incidence and duration of diarrhea. Loss of MyD88 also impaired humoral immunity to RV. Specifically, MyD88 knockout generated less RV-specific IgA and exhibited profoundly reduced RV-specific IgG2c/IgG1 ratios suggesting that MyD88 signaling drives RV-induced Th1 responses. A study of MyD88 bone marrow chimeras indicated that MyD88-dependent control of acute RV infection was mediated by both hemopoietic and non-hemopoietic cells, while generation of RV-specific humoral immunity was driven by MyD88 signaling in hemopoietic cells, which reflected the loss of IL-1β and IL-18 expression by these cells. Thus, TLR signaling and inflammasome cytokines drive innate and adaptive immunity to RV.

22 T-cell Receptor β (TCR β) Complementarity-Determining Region 3 (CDR3) Sequencing Provides a Distinct Profile Defining IBD Patients and Identifies Unique TCR β CDR3 Regions Significantly Associated With Crohn's Disease or Ulcerative Colitis

Background: Rotavirus (RV), the world's leading cause of severe viral gastroenteritis amongst children, primarily infects small intestinal epithelial cells (IECs) and is responsible for over 500,000 deaths and millions of physician visits/hospitalizations per year. While successful resolution of infection and protection against future infection is mediated by adaptive immunity, particularly mucosal IgA, relatively little is known how innate immunity contains acute infection and drives the adaptive immune response to this virus. In light of the broad role of MyD88 in signaling by TLRs and inflammasome cytokine receptors signaling, our goal was examine the consequence of absence of MyD88 in a mouse model of RV infection as means to mechanistically explore innate immunity to this virus. Methods: Suckling and adult mice (WT, MyD88KO, Caspase-1KO, and various bone-marrow irradiation chimeras) mice were inoculated with mouse-passaged RVEC strain. Infection was assessed by ELISA and qRT-PCR. RV replication was assessed by quantitating + to strand ratios. Diarrhea was scored visually. RV-specific immune responses were measured by fecal/serum ELISA. Results: Loss of MyD88 markedly impaired innate immunity to RV resulting in higher levels of virus in feces, intestinal lysates, and peripheral blood cells as well as increased RV replicative ability. Such increased RV levels correlated with a marked increase in the incidence and duration of diarrhea in neonatal mice. These results were fully phenocopied by loss of Caspase-1 and partially mimicked by loss of IL-1 and IL-18 suggesting a role for inflammasome signaling in innate control of RV infection. Such inability to control RV was not observed in irradiated WTmice reconstituted with MyD88KO or Caspase-1KO bone marrow nor could WT bone marrow restore innate immunity to RV in MyD88KO or Caspase-1KO mice suggesting a prominent role for inflammasome signaling in IEC in limiting RV infection/ pathogenesis. Loss of MyD88 also profoundly impaired adaptive immunity to RV. Specifically, MyD88KOmice produced less serum RV-specific IgG and IgA and have Th2 skewed antibody responses indicative of improper Th responses and isotype class switching. In contrast to the role of MyD88 in innate immunity, MyD88-mediated adaptive immunity was independent of Caspase-1, IL-1, and IL-18 and was mediated by MyD88 in bone marrow derived cells. Conclusion: Inflammasome signaling, in IECs, plays a major role in limiting RV infection and, consequently, the diarrheal disease that the great mortality/morbidity caused by this pathogen. TLR signaling in bone-marrow derived cells is important for driving the humoral immune responses that provide lasting protection against this pathogen.

21 MyD88 Is Important for Rotavirus Infection Containment and Specific Adaptive Responses

Background: Rotavirus (RV), the world's leading cause of severe viral gastroenteritis amongst children, primarily infects small intestinal epithelial cells (IECs) and is responsible for over 500,000 deaths and millions of physician visits/hospitalizations per year. While successful resolution of infection and protection against future infection is mediated by adaptive immunity, particularly mucosal IgA, relatively little is known how innate immunity contains acute infection and drives the adaptive immune response to this virus. In light of the broad role of MyD88 in signaling by TLRs and inflammasome cytokine receptors signaling, our goal was examine the consequence of absence of MyD88 in a mouse model of RV infection as means to mechanistically explore innate immunity to this virus. Methods: Suckling and adult mice (WT, MyD88KO, Caspase-1KO, and various bone-marrow irradiation chimeras) mice were inoculated with mouse-passaged RVEC strain. Infection was assessed by ELISA and qRT-PCR. RV replication was assessed by quantitating + to strand ratios. Diarrhea was scored visually. RV-specific immune responses were measured by fecal/serum ELISA. Results: Loss of MyD88 markedly impaired innate immunity to RV resulting in higher levels of virus in feces, intestinal lysates, and peripheral blood cells as well as increased RV replicative ability. Such increased RV levels correlated with a marked increase in the incidence and duration of diarrhea in neonatal mice. These results were fully phenocopied by loss of Caspase-1 and partially mimicked by loss of IL-1 and IL-18 suggesting a role for inflammasome signaling in innate control of RV infection. Such inability to control RV was not observed in irradiated WTmice reconstituted with MyD88KO or Caspase-1KO bone marrow nor could WT bone marrow restore innate immunity to RV in MyD88KO or Caspase-1KO mice suggesting a prominent role for inflammasome signaling in IEC in limiting RV infection/ pathogenesis. Loss of MyD88 also profoundly impaired adaptive immunity to RV. Specifically, MyD88KOmice produced less serum RV-specific IgG and IgA and have Th2 skewed antibody responses indicative of improper Th responses and isotype class switching. In contrast to the role of MyD88 in innate immunity, MyD88-mediated adaptive immunity was independent of Caspase-1, IL-1, and IL-18 and was mediated by MyD88 in bone marrow derived cells. Conclusion: Inflammasome signaling, in IECs, plays a major role in limiting RV infection and, consequently, the diarrheal disease that the great mortality/morbidity caused by this pathogen. TLR signaling in bone-marrow derived cells is important for driving the humoral immune responses that provide lasting protection against this pathogen.