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Title: TLR4, TRIF, and MyD88 are essential for myelopoiesis and CD11c+ adipose tissue macrophage production in obese mice Running title: TLR4, TRIF and MyD88 in Obesity-Induced Inflammation

Obesity induced chronic inflammation is associated with metabolic disease. Results from mouse models utilizing a high-fat diet (HFD) have indicated that an increase in activated macrophages, including CD11c+ adipose tissue macrophages (ATMs), contributes to insulin resistance. Obesity primes myeloid cell production from hematopoietic stem cells (HSCs), and toll-like receptor 4 (TLR4) and the downstream TIR domain–containing adapter protein–inducing interferon-β (TRIF)and MyD88-mediated pathways regulate production of similar myeloid cells after lipopolysaccharide stimulation. However, the role of these pathways in HFD-induced myelopoiesis are unknown. We hypothesized that saturated fatty acids and HFD alter myelopoiesis by activating TLR4 pathways in HSCs, differentially producing proinflammatory CD11c+ myeloid cells that contribute to obesity-induced metabolic disease. Results from reciprocal bone marrow transplants (BMTs) with Tlr4-/and wildtype (WT) mice indicated that TLR4 is required for HFDinduced myelopoiesis and production of CD11c+ ATMs. Experiments with homozygous knockouts of Irakm (encoding a suppressor of MyD88 inactivation) and Trif in competitive BMTs revealed that MyD88 is required for HFD expansion of granulocyte monocyte progenitors and that Trif is required for pregranulocyte macrophage progenitor expansion. A comparison of WT, Tlr4-/-, Myd88-/-, and Trif-/mice on HFD demonstrated that TLR4 plays a role in the production of CD11c+ ATMs, and both Myd88-/and Trif-/mice produced fewer ATMs than WT mice. Moreover, HFD-induced TLR4 activation inhibited macrophage proliferation, leading to greater accumulation of recruited CD11c+ ATMs. Our results indicate that HFD potentiates TLR4 and both its MyD88 and TRIF mediated downstream pathways within progenitors and adipose tissue and leads to macrophage polarization. Introduction: Three decades of research has established the association among obesity, inflammation and metabolic disease (1). Many of the associated co-morbidities of over-nutrition in obese individuals (including insulin resistance, type 2 diabetes (DM), atherosclerosis, cardiovascular disease (CVD) and some cancers) have been attributed to chronic low-grade inflammation, also known as meta-inflammation. More specifically, these obesity-induced diseases have been strongly correlated with an increase in myeloid leukocyte populations (macrophages and neutrophils) (2). Obesity-induced alterations of tissue inflammatory macrophages, both in number and in activation state, have been directly associated with tissue dysfunction. While local macrophage proliferation contributes to this accumulation (3), circulating pro-inflammatory Ly6chi monocyte populations are an important source of tissue macrophages (4). Ly6chi monocytes polarize and traffic into adipose tissue, giving rise to CD11c+ macrophages, specifically in the visceral white adipose tissue (WAT) of obese individuals (5). CD11c+ adipose tissue macrophages (ATMs) are distinguished by a CD64+/CD11c+ phenotype in murine models and a CD206+/CD11c+ phenotype in humans and are recruited through chemokines and adipocyte signals that promote chemotaxis of monocytes (6-11). CD11c+ ATMs accumulate disproportionately in visceral WAT and form ring structures known as crown-like structures (CLS) around damaged adipocytes in obese individuals. In contrast, CD11c-, antiinflammatory resident macrophages predominate in lean individuals (4). The presence of CLS is indicative of a pro-inflammatory state, as these macrophages have been found to secrete cytokines such as TNFα and IL1β (12) and are thus important contributors to metabolic syndrome, specifically in visceral adipose tissue of males (13). Given that the associated cytokines produced by ATMs mimic the inflammation from lipopolysaccharide (LPS) stimulation (14,15), the toll-like receptor 4 (TLR4) pathway has been implicated in obesity-induced inflammation. TLR complexes recognize pathogen-associated molecular patterns (PAMPs) and either signal via a MyD88 dependent or independent pathway (12). Downstream of TLR4, the MYD88 pathway activates the early phase of NFkB, while the MYD88 independent or TRIF pathway 2 by gest on July 5, 2018 hp://w w w .jb.org/ D ow nladed from activates interferon (IFN)-regulatory factor (IRF3) and the late phase of NFkB activation (16). NFkB activation plays a key role in the regulation of both innate and adaptive immunity and consequently has been the target for many anti-inflammatory interventions. The role of TLR4 in the context of diet-induced adipose tissue inflammation and metabolic dysfunction is still unclear. Some investigators have reported a metabolic and inflammatory role for TLR4 (17,18), while others have reported that TLR4 knockout does not improve metabolic function (19). The relative contribution of TRIF and MyD88 pathways to TLR4 mediated obesity induced metabolic impairments and inflammation is unknown. While it has been shown that in obesity TLR4 deficiency significantly promotes alternative macrophage activation (19) and adipose tissue fibrosis (20), recent work in our laboratory demonstrated an additional role for TLR4 in the expansion of hematopoietic stem cells (HSCs) in obesity (2) and a role for MyD88 in the generation of myeloid progenitors in obesity. TLR4 activation in differentiated ATMs with fatty acids (FA)(17) in a high-fat diet environment is one possible mechanism explaining macrophage polarization, but a second mechanism of fatty acid activation within the hematopoietic progenitor cells likely exists as well. There is a paucity of information on the possible dual role for TLR4, TRIF and MyD88 pathways in hematopoietic precursors and in mature tissue macrophages. Therefore, we chose to investigate these pathways together in the context of diet-induced obesity. This is critical to understand as interventions on these inflammatory pathways likely play a role in both the generation of myeloid cells and the activation profile of macrophages in obesity. Based on the literature and prior studies, we sought to evaluate the hypothesis that TLR4, TRIF, and MyD88 are required for hematopoietic stem and progenitor cell (HSPC) responses and tissue macrophage activation with obesity. Studies were carried out using hematopoietic knockout models and in-vivo and in-vitro assays of progenitor and macrophage activation to determine the contribution of TLR4, TRIF and MyD88 pathways to dietinduced inflammation. We demonstrate that these pathways contribute significantly to production of CD11c+ ATMs and myelopoiesis during HFD challenge.