Abstract
Interaction of Cationic Lipid Vaccines With Cells of the Adaptive Immune System
Lisa M. McEwen, PhD
University of Pittsburgh, 2009
When the cationic lipid DOTAP (1,2-dioleyltrimethylammoniumpropane) is used to encapsulate an antigenic peptide from the human papillomavirus E7 oncogene (E7), the resultant DOTAP/E7 particles act as a therapeutic vaccine to cause tumor regression through an antigen-specific immune response when the vaccine is injected into mice bearing E7-positive TC-1 tumors. Of critical importance, the DOTAP works as both a delivery vehicle and an adjuvant without induction of a proinflammatory cytokine response in vivo. It is hypothesized the antigen-specific immune response is mediated by dendritic cells in vivo. To that end, the interaction of murine bone marrow-derived dendritic cells (BMDC) with the vaccine in vitro was investigated. When BMDC were incubated in the presence of DOTAP or the DOTAP/E7 vaccine, there was a dose- and time-dependent up-regulation of co-stimulatory molecule expression, indicating that BMDC were activated by the cationic lipid DOTAP. Further experiments indicated that BMDC were capable of internalizing DOTAP liposomes through many endocytic routes and the vaccine trafficked through the vacuolar pathway. An indirect method was used to validate antigen presentation by BMDC, wherein the generation of antigen-specific effector cells after incubation of CD8+ T lymphocytes, purified from the spleens of naïve mice, with fixed dendritic cells that had been activated by the vaccine, was examined. Not surprisingly, the DOTAP/E7 therapeutic vaccine was capable of initiating the generation of effector CD8+ T lymphocytes in vitro through a conventional mechanism, which requires dendritic cell activation and presentation of the peptide antigen. Interestingly, it was found that the same simple vaccine was capable of generating antigen-specific effectors in vitro in the absence of antigen-presenting cells in a novel pathway of effector generation, based on the expression of CD8 on the cell-surface, T-cell receptors that recognize a specific peptide antigen/MHC complex as well as an antigen-specific increase in IFN-g production. Further studies revealed a possible mechanism for this novel pathway. Taken as a whole, these observations may lead to additional applications of DOTAP both in vitro and in vivo to modulate the immune response toward the correction of a variety of diseases.
Interaction Between Dendritic Cells and Campylobacter jejuni:Role of Toll-Like Receptor Signaling
Vijay Karuppannan Rathinam, PhD
Michigan State University, 2009
Campylobacter jejuni is a clinically significant food-borne pathogen that causes enteritis. Dendritic cells (DCs) are central to initiating immune responses to pathogens. One objective of this study was to understand the interaction of murine DCs with C. jejuni and its impact on induction of T-cell responses mediating resistance. Following infection with C. jejuni, DCs were found to efficiently kill C. jejuni and undergo activation by up-regulating the surface expression of maturation markers and by secreting IL-12, IL-6, and TNF-a. Notably, C. jejuni-infected DCs induced Th1 differentiation of naïve CD4+ T cells. Next, we investigated the role of Toll-like receptor (TLR) signaling in mediating these responses. Up-regulation of maturation markers was significantly impaired in both TLR-2−/− and TLR-4−/− DCs relative to wild-type DCs after C. jejuni challenge. In contrast, TLR-4 deficiency, but not TLR-2 deficiency, profoundly impaired the cytokine responses following C. jejuni infection. Because TLR-4 utilizes both MyD88 and TRIF adapters for signal transduction, we investigated the role of MyD88 and TRIF in these responses. The expression of maturation markers and cytokines in response to C. jejuni was greatly reduced in the absence of either MyD88 or TRIF. Furthermore, C. jejuni infection induced IRF-3 phosphorylation and IFN-b secretion by DCs in a TLR-4-TRIF-dependent fashion, further demonstrating activation of this pathway by C. jejuni. Importantly, TLR-2, TLR-4, MyD88, and TRIF deficiencies all markedly impaired Th1-priming ability of C. jejuni-infected DCs. Thus, our results show for the first time that cooperative signaling through MyD88-dependent and -independent (TRIF) arms of the TLR-4 signaling represents a novel mechanism mediating C. jejuni-induced inflammatory activation of DCs.
Mechanisms of Virus-Induced Type I Diabetes Mellitus
Wieke de Graaf, PhD
Saint Louis University, 2009
Type I diabetes mellitus is an autoimmune disease that is characterized by selective destruction of insulin-secreting beta-cells located in the pancreatic islets of Langerhans. Virus infection has been proposed as an environmental factor that initiates autoimmune-mediated beta-cell damage. Multiple cell types and virus-induced cytokines may contribute to the development of diabetes.
Macrophages have been suggested as important players in diabetes development. In response to virus infection, macrophages produce inflammatory mediators, including interleukin-1 (IL-1) and nitric oxide, which are toxic to beta-cells. The mechanisms of macrophage activation in response to virus infection are incompletely understood. In these studies, we examined the roles of phosphatidylinositol 3-kinase (PI3K) and Src tyrosine kinase in the regulation of macrophage responses to virus infection. We provide evidence that PI3K and Src regulate the inflammatory response of macrophages to encephalomyocarditis virus (EMCV) infection.
Several human and animal studies have implicated type I interferons (IFNs), which are antiviral cytokines, in the development of autoimmune diabetes. The mechanism by which type I IFNs contribute to the pathophysiology of diabetes is not well understood. We propose that type I IFNs contribute to the development of autoimmune diabetes by changing antigen processing in beta-cells. The proteasome plays a key role in antigen processing, by generating peptides that are presented at the cell surface by MHC-I molecules. IFN-gamma is known to alter the composition of the proteasome by inducing the immunoproteasome–PA28 complex, which results in the generation of a different repertoire of presented epitopes at the cell surface. A similar role has only recently been suggested for type I IFNs, based on experiments with hepatocytes. We show that type I IFNs indeed increase the expression of the immunoproteasome subunits and proteasome activator PA28 in pancreatic beta-cells. These results support the emerging view that type I IFNs play a role in the induction of the immunoproteasome–PA28 complex and may underline a novel regulatory pathway that contributes to the development of autoimmune diabetes.
Molecular Insights Into Modulation of Host Innate Immune Response by Viral Proteins of RNA Viruses SARS-CoV, HCV, and Influenza Virus
Santhana Thangavelu Devaraj, PhD
The University of Texas Medical Branch Graduate School of Biomedical Sciences, 2009
One of the first and fast line of defense launched by mammalian hosts to counter virus infection is production of type I interferon (IFN), an innate immune response that generates antiviral state to prevent virus replication and spread by expressing several IFN-stimulated genes. Type I interferon response depends on a set of germ line-encoded receptors called pattern recognition receptors (PRRs) that initiate antiviral signaling upon recognizing distinct pathogen-associated molecular patterns (PAMPs). TLR-3, RIG-I, and MDA-5 trigger complex intertwined signaling pathways in response to viral dsRNA leading to the activation of interferon regulatory transcription factors IRF-3, IRF-7, and NF-κB. These transcription factors mediate inflammatory process to clear virus infection. Viruses can evade host antiviral defenses by using several strategies. SARS coronavirus (SARS-CoV), a highly contagious causative agent of severe acute respiratory syndrome, does not induce interferon response suggesting an unknown immune evasive mechanism. My experiments demonstrate that papain-like protease (PLpro) encoded by SARS-CoV is a potent interferon antagonist that functions independent of its protease activity. PLpro directly interacts with IRF-3 preventing its phosphorylation, dimerization, nuclear translocation and thus inhibits type I interferon response triggered by TLR-3/RIG-I pathways. Hepatitis C virus is a major blood-borne pathogen responsible for 100,000 deaths worldwide annually due to chronic liver cirrhosis. In cell culture, normal human hepatocytes are not permissive to HCV replication due to intact TLR-3/RIG-I/MDA-5 antiviral signaling pathways. However, human hepatoma cells defective in antiviral signal pathways are found to permit HCV replication. My experiments involving reconstitution of functional TLR-3 signaling pathways in human heptoma cells demonstrate that TLR-3 plays a major role in HCV cellular permissiveness. Finally, my studies with influenza virus NS1 protein demonstrate that NS1 antagonizes the IFN response by blocking RIG-I activation in a strain-specific manner. In conclusion, I have made an attempt to understand the complex antiviral signaling pathways at the cellular level in context to 3 distinct single-strand RNA viruses namely, SARS-CoV, HCV, and influenza virus. Though, these viruses are detected by the same set of PRRs to trigger antiviral signaling, the mechanism by which they evade antiviral response appears to be distinct.
Novel Roles for Chemokines in Acute Cardiac Allograft Rejection
Joshua Michael Rosenblum, PhD
Case Western Reserve University, 2009
Chemokines play varied roles in innate and adaptive immune responses, both in states of health and in pathological processes. In solid organ transplantation, acute allograft rejection is primarily mediated by donor-reactive T cells that have been primed in the recipient spleen and then migrate to the graft where they release effector cytokines that mediate cell death and tissue destruction. During this effector phase of the alloresponse, the C-X-C-motif (CXC) chemokines that bind CXCR3 play an important role in recruiting primed effector T cells to the allograft. To a less significant extent, the CCR5-binding chemokines also function in this recruitment process; although in models requiring regulation of the alloresponse by regulatory T cells (Tregs), there is evidence that CCR5 may be important for the function of Tregs.
In Chapter 2 of this dissertation, we demonstrate that CCR5-deficient recipients of single class II MHC-mismatched cardiac allografts produce an exaggerated and prolonged alloresponse as compared to wild-type recipients. Acute rejection of the grafts is accompanied by a significantly decreased number of FoxP3+ Tregs in the allografts and increased numbers of effector CD4 T cells. Adoptive transfer of wild-type CD4+ CD25+ Tregs to CCR5−/− recipients restores graft survival. Chapters 3 and 4 directly test the role of CXCR3 and CXCL9 in acute rejection of full MHC-mismatched cardiac allografts. We show that blockade of CXCR3 in wild-type allograft recipients prolongs graft survival, causes a decrease in the number of donor-reactive, IFN-g-producing CD8 T cells, but does not affect T-cell infiltration into the grafts. In CXCL9−/− allograft combinations, the frequency of donor-reactive, IFN-g-producing T cells is also significantly reduced. Natural killer cells produced IFN-g in the allograft recipient spleen within 24–48 h posttransplant and this induces early CXCL9 production. Taken together, these results indicate that CXCR3 and CCR5 function in the priming and regulatory processes, respectively, and that these roles must be considered prior to any therapeutic interventions using blocking agents to prolong graft survival.
Opposing Roles of Interferon Gamma in Transplantation Under Co-Stimulation Blockade
Shana Coley, PhD
Emory University, 2009
During the last 50 years, organ transplantation has become a favored treatment option for patients with otherwise incurable conditions. Nevertheless, the immune response to donor tissue remains the major obstacle to long-term graft survival. Prolongation of allograft survival through T-cell co-stimulation blockade (CoB) is based on the principle that T-cell receptor stimulation in the absence of co-stimulation leads to abortive activation or deletion of T cells. However, resistance to CoB-induced allograft survival is seen in some mouse models. CD8 T cells are necessary for this resistance, but the mechanism by which CD8 T cells mediate rejection in the absence of major co-stimulatory signals is poorly understood. Interferon-gamma (IFN-g) promotes CD8 T-cell responses, but IFN-g-deficient mice show early graft loss despite CoB. In contrast, we found that IFN-g receptor-deficient mice show dramatically prolonged graft survival under CoB. Investigating this paradox, we addressed the effects of IFN-g on T-cell alloresponses in vivo independent of the effects of IFN-g on graft survival. We found that neither IFN-g receptor-deficient recipients nor IFN-g-deficient recipients mount anti-graft CD8 T-cell responses. To explain graft loss despite undetectable T-cell responses in IFN-g-deficient recipients, we found that direct action of IFN-g on the graft was necessary for graft survival, as either IFN-g neutralization in IFN-g receptor-deficient recipients or the lack of the IFN-g receptor on the graft precipitated early graft loss. Furthermore, IFN-g decreased cellular infiltrates and hemorrhage and increased transcripts for tolerance-associated molecules within grafts at early time points. In contrast to other models, NK cells were dispensable but CD4+ cells were necessary for graft maintenance. At late time points, IFN-g held in check a dormant immune response, as IFN-g neutralization not only precipitated graft loss but also led to increased transcripts for cell-mediated response genes within graft-draining lymph nodes. Importantly, late-term control via IFN-g was by direct action on graft-derived cells and by indirect action on the recipient immune system. Thus, IFN-g is required both for the recipient to mount a donor-specific CD8 T-cell response under CoB as well as for allografts to initiate and maintain survival after transplantation.
Regulation of Neuronal CXCR4 Signaling by Opioids
Rajarshi Sengupta, PhD
Drexel University College of Medicine, 2009
Heterologous desensitization is a potential mechanism involved in the regulation of chemokine receptors. The chemokine CXCL12 and its receptor CXCR4, also a HIV co-receptor, play a significant role in neuronal development and homeostasis. Studies from our lab have shown that mu-opioid agonists, such as DAMGO, and the endogenous peptide endomorphin-1 inhibit the neuroprotective action of CXCL12 in cultured rat cortical fleurons. Here, we studied the cellular and molecular mechanisms behind opiate action and identified a novel mediator of the effect of opioids on CXCR4 signaling in vivo. In brief, we show that the inhibitory action of opioids is due to a direct effect on neurons (ie, flot glia-mediated) and requires prolonged mu-opioid receptor (MOR) stimulation. CXCR4-induced Gai and Gbg activities are both suppressed following DAMGO treatment as indicated by analysis of different downstream targets regulated by CXCL12. GTPgS incorporation assays in brain also indicate a reduced coupling of CXCR4 to Gai in the cortex and hippocampus of morphine-treated rats. Furthermore, morphine pretreatment inhibits prosurvival signaling in cortical cultures. In addition, treatment of cultured neurons with morphine or DAMGO also leads to a decrease in the ligand-induced CXCR4 phosphorylation. Overall these findings point to a deficit of CXCR4 activation in opioid-treated neurons. Interestingly, initial studies with brain tissue samples from HIV and control patients show a reduction of CXCR4 phosphorylation (pS339) in HIV+ patients with neurological problems as compared to HIV− control and neurologically normal HIV+ individuals, suggesting that impairment in CXCR4 may lead to neuronal dysfunction in humans. Finally, we demonstrate for the first time the involvement of the ubiquitous peptide ferritin heavy chain (FHC) in mediating opioids’ action on CXCR4 signaling. Treatment of cortical neurons with DAMGO or morphine up-regulates FHC levels through activation of MOR. Elevated FHC levels are also detected in cortical tissue from rats treated with morphine. The FHC increase temporarily correlates with the reduction of CXCR4-mediated responses whereas inhibition of FHC expression by siRNA prevents the effect of opioids on CXCR4. Altogether, these data suggest that opiates interfere with normal CXCR4 function in the brain and may exacerbate progression to neuroAIDS in HIV-positive drug users.
Role and Importance of NS1 Protein of Avian Influenza Virus to Grow in the Presence of Interferon and Evaluation of the NS1 Mutant Viruses as Potential DIVA Vaccines
Vinayak R. Brahmakshatriya, PhD
Texas A&M University, 2009
A proper vaccination program can play a critical role in prevention and control of avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses (LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to trade implications, differentiation of infected from vaccinated animals (DIVA) is an important issue in the control of AI. Therefore, the development and characterization of vaccine candidates with DIVA properties is critical in improving vaccination programs. Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a major role in blocking the host’s antiviral response. Using an 8-plasmid reverse genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant (H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3 and H5N3/NS1/144 were compared in cell culture and in different age embryonated chicken eggs. Our results confirmed that NS1 is involved in down-regulation of interferon as shown by IFN-b mRNA expression analysis and by the inability of H5N3/NS1-144 to efficiently grow in older age, interferon-competent, chicken embryos. However with regards to safety, the virus reverted to virulence within 5 back passages in chickens and was therefore not a safe vaccine candidate. However, the killed form of H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection not significantly different from the parental H5N3 as vaccine. To further understand the reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1 gene for the presence of mutations. RT-PCR and sequence analysis of the NS gene in all 3 lineages showed the presence of a 54-amino acid deletion resulting in the generation of a 87-amino acid long NS1 ORF with a point mutation (L80V) at the site of deletion. In addition, the NS1 ORF in lineages L2 and L3 presented 2 additional point mutations in the RNA-binding domain (Q40R and T73M). To determine if these mutations played a role in increased virulence, recombinant viruses expressing these mutant NS1 proteins in the background of parental virus were generated by reverse genetics and their replication properties and pathogenicity was examined in vitro, in ovo, and in vivo systems.
Our results showed that the 87-amino acid long NS1 protein clearly increased virus replication and virulence specifically in interferon-competent systems. In addition, the 2-point mutations in the RNA-binding domain of NS1 ORF expressing 87-amino acid protein slightly increased the virus virulence.
Overall, this study reinforces the role of NS1 in influenza virus pathogenicity and supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA vaccines.
The Influence of Cell Type on Alphavirus Type I Interferon Induction
Crystal W. Burke, PhD
Louisiana State University Health Sciences Center, Shreveport, 2009
Important veterinary and human pathogens, alphaviruses are positive-sense single-stranded RNA viruses comprised of 2 groups: the Old World alphaviruses (eg, Sindbis virus [SINV] and chikungunya virus [CHIKV]) and the New World alphaviruses (eg, Venezuelan [VEEV] and eastern [EEEV] equine encephalitis virus). Because sensitivity of these viruses to the effects of type I interferon (IFN-a/b) correlates with the severity of disease in vivo, IFN-a/b is a critical factor in disease survival. However, little is understood about the host pattern recognition receptors (PRRs), downstream signaling molecules, or cell types involved in induction of IFN-a/b. Infection of murine fibroblasts (MEFs) with wild-type (wt) alphaviruses did not stimulate production of IFN-a/R despite the fact that wt SINV or VEEV infection resulted in activation and translocation of interferon regulatory factor 3 (IRF-3) to the nucleus. In contrast, a non-cytopathic SINV (39nc) defective in shut-off of host macromolecular synthesis induced IFN-a/b production at late times postinfection and was dependent upon the cytoplasmic kinase TBK-1. Examination of PRRs revealed that IFN-a/b production by 39nc was dependent on the RNA-binding proteins MDA-5 and PKR, but not RIG-I. We hypothesize that wt alphaviruses do not specifically block IFN-a/b induction pathways but, instead, may inhibit IFN-a/b production through inhibition of host macromolecular synthesis.
Although wt alphavirus infection of MEFs did not result in IFN-a/b production, IFN-a/b could be detected in the serum of infected mice as early as 8–12 hpi suggesting that a different type of cell produced IFN-a/b in vivo. Examination of conventional dendritic cells revealed that these cells were not responsible for the early systemic IFN-a/b but, instead, cells in plasmacytoid DC (pDC) cultures and immature macrophage (IM) cultures produced early IFN-a/b. SINV infection of pDC cultures activated pDCs, but purified pDCs were nonpermissive to infection and failed to produce detectable IFN-a/b. Therefore, another cell in the heterogeneous cultures was likely responsible. IM cultures infected with SINV produced IFN-a/b in a Toll-like receptor-independent but IRF-dependent manner. The SINV-infected cells within pDC cultures, IMCs, and the popliteal lymph node of infected mice were CD11c− CD11b+, indicating a previously unappreciated role of immature macrophage-like cells as important IFN-a/b producers after alphavirus infection.
The Role of T Cell-Associated Polarizing Transcription Factors inDendritic Cell Priming of T Cells Toward Immunity or Tolerance;Role of T-bet or Foxp3 Ectopic Expression in Dendritic Cells
Michael Wheeler Lipscomb, PhD
University of Pittsburgh, 2009
Dendritic cells (DC) are professional antigen-presenting cells that can prime naïve T cells to elicit immunity or tolerance. The ability to regulate immunity or tolerance is governed by the “type” of polarization state of activated T cells. T-bet has been identified as the master regulator of Type 1 polarization in T cells, and its expression is essential for immunity. Interestingly, T-bet is also expressed in DC and its abolishment has been shown to impair Type 1 T-cell responses in limited studies. Conversely, Foxp3 expression in T-regulatory cells engenders a tolerogenic phenotype that can suppress T-cell responses (as well as DC induction of immunity). Foxp3 expression in non-T-cell subsets, such as adenocarinoma, has also shown immunosuppressive characteristics in the tumor microenvironment and draining lymph nodes. Therefore, I examined the role of T-bet or Foxp3 expression in the DC in modulating T-cell responses. T cells were primed with T-bet expressing DC (DC.T-bet) or Foxp3 expressing DC (DC.Foxp3) and responses were thoroughly investigated. DC.T-bet potently primed naïve T cells toward Type 1 immunity, inducing 2–3-fold increased levels of T-bet, IFN-g, CXCR3, and granzyme B. Little-to-no changes were found in DC co-stimulatory molecule expression; however, DC were completely impaired in production of proinflammatory and Type 1-inducing cytokines. We confirmed cytokine-independent Type 1 polarization of T cells via neutralization studies. In analogous studies, Foxp3 ectopic expression in DC was found to restrain Type 1 and 17 polarized T-cell responses while concomitantly generate CD4+Foxp3+CD25+ T-regulatory cell subsets that co-expressed high levels of CTLA-4, CD25, NRP-1, and GITR. These in vitro generated T-regulatory cells (by DC.Foxp3 and not control DC) suppressed both naïve and memory CD8+ T-cell proliferation and IFN-g production. Neutralizing agents confirmed that the tryptophan-catabolizing enzyme IDO and the immunosuppressive cytokine TGF-b were partially dependent for both suppressing Type 1 T-cell responses and generating functionally suppressive Tregs. In summation, this work shows that T-bet and Foxp3 expression in DC play similar roles to expression in T cells by governing immunity or tolerance, respectively.
The Roles of STAT6 and STAT4 in Glucose and Lipid Homeostasis
Roberto Ricardo-Gonzalez, PhD
Stanford University, 2009
Innate and adaptive immune responses perform critical functions in host defense, tissue injury, and tissue remodeling. Although these inflammatory responses can be life-saving during times of infection or tissue damage, uncontrolled inflammation contributes to the pathogenesis of various degenerative, autoimmune, and metabolic diseases, including multiple sclerosis, arthritis, atherosclerosis, and type 2 diabetes. For instance, inflammatory activation of macrophages and the release of T-helper 1 (TH1) type inflammatory cytokines like tumor necrosis factor-a, interleukin-6, and interleukin-1b are closely linked to the pathogenesis of obesity and the detrimental sequelae of insulin resistance and metabolic syndrome. In the past, the development of new therapeutic interventions to treat this inflammatory response focused on either blockade of these inflammatory cytokines, or suppression of the downstream signaling pathways that these cytokines activate. However, biasing the immune response toward the TH2 axis was an alternative approach that successfully attenuated TH1-type inflammation in murine models of autoimmunity without the attendant immunosuppressive sequelae. I hypothesized that the anti-inflammatory axis driven by TH2-type responses could ameliorate the detrimental effects of obesity-induced insulin resistance and metabolic syndrome. I approached this hypothesis from 2 independent but complementary angles: (1) identification of transcriptional regulators that promote alternative macrophage activation and (2) disruption (loss of function) and activation (gain of function) of the TH2 axis of inflammation.
During a search for genetic pathways that control alternative macrophage activation, our lab identified the peroxisome proliferator-activated receptor (PPAR)-g, a sensor of fatty acids, as a gene that was markedly induced in macrophages stimulated with interleukin-4 (IL-4). We generated mice with a macrophage-specific deletion of PPAR-g, and showed that PPAR-g was critical for the maturation of alternatively activated macrophages. Disruption of PPAR-g in myeloid cells impaired alternative macrophage activation, and thereby predisposed these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. In addition, we showed that in response to IL-4, PPAR-d directed the expression of the alternative phenotype in liver macrophages (Kupffer cells) of lean and obese mice. Importantly, adoptive transfer of PPAR-d−/− bone marrow into wild-type mice diminished alternative activation of hepatic macrophages, which caused hepatic dysfunction and insulin resistance. Suppression of hepatic oxidative metabolism was recapitulated by co-culturing hepatocytes with PPAR-d−/− macrophages or their conditioned media, which indicated the direct involvement of Kupffer cells in controlling liver lipid metabolism. Altogether, these experiments demonstrate that alternative activation of adipose tissue macrophages and Kupffer cells plays a central role in the maintenance of glucose and lipid homeostasis.
Since it is well established that TH1-type inflammation contributes to the pathogenesis of obesity-induced insulin resistance, I reasoned that biasing the adaptive immune response toward the TH2 axis would mitigate the metabolic sequelae of obesity. Genetic disruption of signal transducer and activator of transcription 6 (STAT6), the mediator of TH2-type immune responses, inhibited insulin action, while administration of the TH2 cytokine interleukin-4 (IL-4) improved glucose tolerance and insulin sensitivity in obese mice. The anti-diabetic effects of the IL-4/STAT6 axis were partially mediated by regulation of hepatic fuel selection via inhibition of peroxisome proliferator-activated receptor-a (PPAR-a).
Since mice unable to activate the TH2 axis of inflammation exhibited marked impairment in nutrient homeostasis, I explored the role of the signal transducer and activator of transcription 4 (STAT4), a key transcriptional regulator of TH1 responses, in glucose and lipid homeostasis. STAT4 null mice were resistant to diet-induced glucose intolerance. Strikingly, STAT4-deficient mice exhibited fasting hypoglycemia, due in part to blunted expression of the transcriptional program associated with the fasting response. Moreover, fasting induced interleukin-12 (IL-12) production by liver Kupffer cells. IL-12 was able to synergize with gluconeogenic factors to increase the expression of genes associated with the fasting response.
Together, these findings identify a new biological function for TH2 and TH1 immunity in the regulation of energy homeostasis. I show that activation of the TH2 inflammatory program ameliorates the harmful effects of obesity, and I identify STAT6 as a key transcriptional regulator of energy homeostasis. Moreover, I show that the TH1 signaling pathway is critical to the initiation of the fasting response. Thus, my thesis work uncovers novel transcriptional cross talk between the immune and metabolic systems, indicating that these programs might have co-evolved to control essential functions in metazoans.