Depletion of B cells rejuvenates the peripheral B‐cell compartment but is insufficient to restore immune competence in aging

Aging is associated with increasing prevalence and severity of infections caused by a decline in bone marrow (BM) lymphopoiesis and reduced B‐cell repertoire diversity. The current study proposes a strategy to enhance immune responsiveness in aged mice and humans, through rejuvenation of the B lineage upon B‐cell depletion. We used hCD20Tg mice to deplete peripheral B cells in old and young mice, analyzing B‐cell subsets, repertoire and cellular functions in vitro, and immune responsiveness in vivo. Additionally, elderly patients, previously treated with rituximab healthy elderly and young individuals, were vaccinated against hepatitis B (HBV) after undergoing a detailed analysis for B‐cell compartments. B‐cell depletion in old mice resulted in rejuvenated B‐cell population that was derived from de novo synthesis in the bone marrow. The rejuvenated B cells exhibited a "young"‐like repertoire and cellular responsiveness to immune stimuli in vitro. Yet, mice treated with B‐cell depletion did not mount enhanced antibody responses to immunization in vivo, nor did they survive longer than control mice in "dirty" environment. Consistent with these results, peripheral B cells from elderly depleted patients showed a "young"‐like repertoire, population dynamics, and cellular responsiveness to stimulus. Nevertheless, the response rate to HBV vaccination was similar between elderly depleted and nondepleted subjects, although antibody titers were higher in depleted patients. This study proposes a proof of principle to rejuvenate the peripheral B‐cell compartment in aging, through B‐cell depletion. Further studies are warranted in order to apply this approach for enhancing humoral immune responsiveness among the elderly population.     

Flagellin‐dependent TLR5/caveolin‐1 as a promising immune activator in immunosenescence

The age‐associated decline of immune responses causes high susceptibility to infections and reduced vaccine efficacy in the elderly. However, the mechanisms underlying age‐related deficits are unclear. Here, we found that the expression and signaling of flagellin (FlaB)‐dependent Toll‐like receptor 5 (TLR5), unlike the other TLRs, were well maintained in old macrophages, similar to young macrophages. The expression and activation of TLR5/MyD88, but not TLR4, were sensitively regulated by the upregulation of caveolin‐1 in old macrophages through direct interaction. This interaction was also confirmed using macrophages from caveolin‐1 or MyD88 knockout mice. Because TLR5 and caveolin‐1 were well expressed in major old tissues including lung, skin, intestine, and spleen, we analyzed in vivo immune responses via a vaccine platform with FlaB as a mucosal adjuvant for the pneumococcal surface protein A (PspA) against Streptococcus pneumoniae infection in young and aged mice. The FlaB‐PspA fusion protein induced a significantly higher level of PspA‐specific IgG and IgA responses and demonstrated a high protective efficacy against a lethal challenge with live S. pneumoniae in aged mice. These results suggest that caveolin‐1/TLR5 signaling plays a key role in age‐associated innate immune responses and that FlaB‐PspA stimulation of TLR5 may be a new strategy for a mucosal vaccine adjuvant against pneumococcal infection in the elderly.     

The natural killer cell dysfunction of aged mice is due to the bone marrow stroma and is not restored by IL‐15/IL‐15Rα treatment

Immune dysfunctions in the elderly result in increased susceptibility to infectious diseases, cancer, and autoimmune diseases. Natural killer (NK) cells are bone marrow‐derived lymphocytes crucial for host defense against several infections and cancer. We have previously shown that compared to young, aged C57BL/6 mice have decreased numbers of mature NK cells in the blood, spleen, and bone marrow, resulting in susceptibility to mousepox, a lethal disease caused by ectromelia virus. Here, we describe further age‐related defects in NK cells including reduced proliferation in vivo, additional signs of immaturity, and dysregulated expression of activating and inhibitory receptors. Aging also alters the expression of collagen‐binding integrins in conventional NK cells and the frequency and phenotype of liver tissue‐resident NK cells. We additionally show that the defect in NK maturation is the consequence of deficient maturational cues provided by bone marrow stromal cells. Moreover, we demonstrate that in aged mice, treatment with complexes of the cytokine IL‐15 and IL‐15Rα induce massive expansion of the NK cells, but most of these NK cells remain immature and are unable to restore resistance to mousepox. The use of rodent model to understand immunosenescence may help the development of treatments to improve the immune fitness of the aged. Our work with NK cells should contribute toward this goal.     

Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle‐derived nitric oxide

Muscle aging is associated with changes in myeloid cell phenotype that may influence age‐related changes in muscle structure. We tested whether preventing age‐related reductions in muscle neuronal nitric oxide synthase (nNOS) would obviate age‐related changes in myeloid cells in muscle. Our findings show that muscle aging is associated with elevations of anti‐inflammatory M2a macrophages that can increase muscle fibrosis. Expression of a muscle‐specific nNOS transgene in mice prevented age‐related increases in M2a macrophages. Transgene expression also reduced expression of collagens and decreased muscle fibrosis. The nNOS transgene prevented age‐related increases in arginase‐1 but did not influence TGFβ expression, indicating that the transgene may prevent age‐related muscle fibrosis by inhibiting the arginase‐dependent profibrotic pathway. Although aged satellite cells or fibro‐adipogenic precursor (FAPs) cells also promote fibrosis, transgene expression had no effect on the expression of key signaling molecules that regulate fibrogenic activity of those cells. Finally, we tested whether increases in M2a macrophages and the associated increase in fibrosis were attributable to aging of myeloid lineage cells. Young bone marrow cells (BMCs) were transplanted into young or old mice, and muscles were collected 8 months later. Muscles of young mice receiving young BMCs showed no effect on M2a macrophage number or collagen accumulation compared to age‐matched, nontransplanted controls. However, muscles of old mice receiving young BMCs showed fewer M2a macrophages and less accumulation of collagen. Thus, the age‐related increase in M2a macrophages in aging muscle and the associated muscle fibrosis are determined in part by the age of bone marrow cells.     

Sclerostin is expressed in osteoclasts from aged mice and reduces osteoclast‐mediated stimulation of mineralization

Osteoclast‐mediated bone resorption precedes osteoblast‐mediated bone formation through early adulthood, but formation fails to keep pace with resorption during aging. We previously identified several factors produced by osteoclasts that promote bone formation. In this study, we determined if osteoclast‐produced factors contribute to the impaired bone formation with aging. We previously found that mice between the ages of 18 and 22 months develop age‐related bone loss. Bone marrow‐derived pre‐osteoclasts were isolated from 6‐week, 12‐month, and 18‐ to 24‐month‐old mice and differentiated into osteoclasts in vitro. Conditioned media were collected and compared for osteoblast mineralization support. Conditioned medium from osteoclasts from all ages was able to support mineralization of bone marrow stromal cells. Concentrating the conditioned medium from 6‐week‐old and 12‐month‐old mouse marrow cells‐derived osteoclasts enhanced mineralization support whereas concentrated conditioned medium from 18‐ to 24‐month‐old mouse marrow‐derived osteoclasts repressed mineralization compared to base medium. This observation suggests that an inhibitor of mineralization was secreted by aged murine osteoclasts. Gene and protein analysis revealed that the Wnt antagonist sclerostin was significantly elevated in the conditioned media from 24‐month‐old mouse cells compared to 6‐week‐old mouse cells. Antibodies directed to sclerostin neutralized the influences of the aged mouse cell concentrated conditioned media on mineralization. Sclerostin is primarily produced by osteocytes in young animals. This study demonstrates that osteoclasts from aged mice also produce sclerostin in quantities that may contribute to the age‐related impairment in bone formation. J. Cell. Biochem. 114: 1901–1907, 2013. © 2013 Wiley Periodicals, Inc.     

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes

Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone marrow, their characteristics differ on the compartment from which they are derived. In this work, we investigated the contribution of phagocytosis for tumor necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently, depending on their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors was sufficient for a maximal production of TNF and interleukin-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating the different actors of signalization, we found that p38 kinase and phosphatidylinositol 3-kinase were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α(5)β(1)-integrin significantly decreased TNF production in response to HKSA in all three cell types. Finally, using mononuclear phagocytes from NOD2 knockout mice, we observed that TNF production in response to HKSA was dependent on NOD2 for monocytes and peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways. The influence of the compartments on cell properties and behavior should be taken into account, to better understand cell physiology and host-pathogen interaction, and to define efficient strategies to fight infection.     

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63⁺ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.     

Targeting macrophages rescues age‐related immune deficiencies in C57BL/6J geriatric mice

Changes to innate cells, such as macrophages and myeloid‐derived suppressor cells (MDSCs), during aging in healthy or tumor‐bearing hosts are not well understood. We compared macrophage subpopulations and MDSCs from healthy young (6–8 weeks) C57BL/6J mice to those from healthy geriatric (24–28 months) mice. Spleens, lymph nodes, and bone marrow of geriatric hosts contained significantly more M2 macrophages and MDSCs than their younger counterparts. Peritoneal macrophages from geriatric, but not young, mice co‐expressed CD40 and CX3CR1 that are usually mutually exclusively expressed by M1 or M2 macrophages. Nonetheless, macrophages from geriatric mice responded to M1 or M2 stimuli similarly to macrophages from young mice, although they secreted higher levels of TGF‐β in response to IL‐4. We mimicked conditions that may occur within tumors by exposing macrophages from young vs. geriatric mice to mesothelioma or lung carcinoma tumor cell–derived supernatants. While both supernatants skewed macrophages toward the M2‐phenotype regardless of age, only geriatric‐derived macrophages produced IL‐4, suggesting a more immunosuppressive tumor microenvironment will be established in the elderly. Both geriatric‐ and young‐derived macrophages induced allogeneic T‐cell proliferation, regardless of the stimuli used, including tumor supernatant. However, only macrophages from young mice induced T‐cell IFN‐γ production. We examined the potential of an IL‐2/agonist anti‐CD40 antibody immunotherapy that eradicates large tumors in young hosts to activate macrophages from geriatric mice. IL‐2‐/CD40‐activated macrophages rescued T‐cell production of IFN‐γ in geriatric mice. Therefore, targeting macrophages with IL‐2/anti‐CD40 antibody may improve innate and T‐cell immunity in aging hosts.     

Osteoclast precursors in bone marrow and peritoneal cavity

Osteoclasts differentiate from cells that share some phenotypes with mature macrophages and monocytes, but early precursors for osteoclasts still remain obscure. To characterize osteoclast precursors, using monoclonal anti-c-Fms and anti-c-Kit antibodies, bone marrow cells were separated and the frequency of clonogenic progenitors were measured. Osteoclast precursors in the bone marrow mainly expressed c-Kit and diminished in frequency when they expressed c-Fms. In contrast to bone marrow, the precursors in the peritoneal cavity were enriched with a population of c-Fms⁺. Injection of these antibodies into mice demonstrated that peritoneal osteoclast precursors were sensitive to anti-c-Fms but not to anti-c-Kit antibodies, whereas those in bone marrow only declined in the presence of both antibodies. Meanwhile, c-Fms as opposed to c-Kit played an essential role in the generation of osteoclasts in cultures. We also compared osteoclast precursors with colony forming cells (CFU-M) by a macrophage colony stimulating factor. CFU-M in bone marrow decreased when anti-c-Kit antibody was administered and no CFU-M was detected in peritoneum. In this study, we show differences between proliferative potential osteoclast precursors maintained in bone marrow and peritoneum and between CFU-M and osteoclast precursors. J. Cell. Physiol. 170:241–247, 1997. © 1997 Wiley-Liss, Inc.     

Macrophage maturation: differences in complement secretion by marrow, monocyte, and tissue macrophages detected with an improved hemolytic plaque assay

In order to examine one function of mononuclear phagocytes during maturation from bone marrow precursors to tissue macrophages, an improved hemolytic plaque assay for the detection of synthesis of the second (C2) and fourth (C4) components of C by single cells was developed. With this method, production of C2 and C4 was assessed in cell populations derived from bone marrow, blood, lung, peritoneum, and spleen. The proportion of cells producing C2 and C4 in each population varied. Approximately 10% of bone marrow cells produced C4, but not detectable C2 plaque-forming cells (PFC) were detected. Circulating monocytes yielded about 10% PFC each for C2 and C4. The proportion of C2-producing cells in tissue macrophages varied from approximately 2% in bronchoalveolar macrophages to about 45% in peritoneal and splenic macrophage populations, whereas C4 production by macrophages from lung, peritoneum, and spleen were all approximately 45%. These data suggest that differences in C biosynthesis characterize mononuclear phagocytes at different stages of maturation.     

In vivo GDF3 administration abrogates aging related muscle regeneration delay following acute sterile injury

Tissue regeneration is a highly coordinated process with sequential events including immune cell infiltration, clearance of damaged tissues, and immune‐supported regrowth of the tissue. Aging has a well‐documented negative impact on this process globally; however, whether changes in immune cells per se are contributing to the decline in the body’s ability to regenerate tissues with aging is not clearly understood. Here, we set out to characterize the dynamics of macrophage infiltration and their functional contribution to muscle regeneration by comparing young and aged animals upon acute sterile injury. Injured muscle of old mice showed markedly elevated number of macrophages, with a predominance for Ly6C^high pro‐inflammatory macrophages and a lower ratio of the Ly6C^low repair macrophages. Of interest, a recently identified repair macrophage‐derived cytokine, growth differentiation factor 3 (GDF3), was markedly downregulated in injured muscle of old relative to young mice. Supplementation of recombinant GDF3 in aged mice ameliorated the inefficient regenerative response. Together, these results uncover a deficiency in the quantity and quality of infiltrating macrophages during aging and suggest that in vivo administration of GDF3 could be an effective therapeutic approach.     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.     

Age‐dependent effect between MARCO and TLR4 on PMMA particle phagocytosis by macrophages

Progressive generation of total joint implant‐derived wear particles is one of the major risk factors in development of peri‐prosthetic osteolysis especially in the aging society. It is commonly accepted that macrophages predominantly drive the inflammatory response to wear debris particles. Among various surface receptors that activate the macrophages to phagocytize particles, it is believed that the Toll‐like receptor 4 (TLR4) and the scavenger macrophage receptor with collagenous structure (MARCO) play key roles in recognition of wear debris particles. However, a strong body of evidence indicates an age‐dependent diminished function of human TLRs. Thus, we hypothesized that the MARCO receptor may be more engaged than TLRs in the phagocytosis of wear debris particles which in turn up‐regulate production of pro‐inflammatory cytokines from aged macrophages. We demonstrated that peritoneal macrophages isolated from aged mice show elevated expression of MARCO receptor compared to that from young mice. In contrast the expression of TLR4 was significantly decreased on the surface of aged macrophages. Furthermore, using anti‐MARCO and anti‐TLR4 neutralizing mAbs, we demonstrated the age‐dependent pathogenic role of MARCO, but not TLR4, receptor in promoting poly‐methyl methacrylate (PMMA) bone cement particles phagocytosis by macrophages leading to the release of pro‐inflammatory cytokines migration inhibitory factor and tumour necrosis factor in vitro. These data also suggest that the approach to neutralize MARCO may lead to the development of therapeutic regimen for the prevention of particle‐induced osteolysis in aged patients.     

Aging aggravated liver ischemia and reperfusion injury by promoting STING‐mediated NLRP3 activation in macrophages

Although aggravated liver injury has been reported in aged livers post‐ischemia and reperfusion (IR), the underlying mechanism of innate immune activation of aged macrophages is not well understood. Here, we investigated whether and how Stimulator of interferon genes (STING) signaling regulated macrophage proinflammatory activation and liver IR injury. Mice were subjected to hepatic IR in vivo. Macrophages isolated from IR‐stressed livers and bone marrow‐derived macrophages (BMDMs) from young and aged mice were used for in vitro studies. Enhanced nucleotide‐binding domain and leucine‐rich repeat containing protein 3 (NLRP3) activation was found in both livers and macrophages of aged mice post‐IR. NLRP3 knockdown in macrophages inhibited intrahepatic inflammation and liver injury in both young and aged mice. Interestingly, enhanced activation of the STING/ TANK‐binding kinase 1 (TBK1) signaling pathway was observed in aged macrophages post‐IR and mitochondria DNA (mtDNA) stimulation. STING suppression blocked over‐activation of NLRP3 signaling and excessive secretion of proinflammatory cytokines/chemokines in the mtDNA‐stimulated BMDMs from aged mice. More importantly, STING knockdown in macrophages abrogated the detrimental role of aging in aggravating liver IR injury and intrahepatic inflammation. Finally, peripheral blood from the recipients undergoing liver transplantation was collected and analyzed. The results showed that the elderly recipients had much higher levels of TNF‐α, IL‐6, IL‐1β, and IL‐18 post‐transplantation, indicating increased NLRP3 activation in lR‐stressed livers of elderly recipients. In summary, our study demonstrated that the STING‐NLRP3 axis was critical for the proinflammatory response of aged macrophages and would be a novel therapeutic target to reduce IR injury in elderly patients.     

Neuropeptide Y regulates the hematopoietic stem cell microenvironment and prevents nerve injury in the bone marrow

Many reports have revealed the importance of the sympathetic nervous system (SNS) in the control of the bone marrow environment. However, the specific role of neuropeptide Y (NPY) in this process has not been systematically studied. Here we show that NPY‐deficient mice have significantly reduced hematopoietic stem cell (HSC) numbers and impaired regeneration in bone marrow due to apoptotic destruction of SNS fibers and/or endothelial cells. Furthermore, pharmacological elevation of NPY prevented bone marrow impairments in a mouse model of chemotherapy‐induced SNS injury, while NPY injection into conditional knockout mice lacking the Y1 receptor in macrophages did not relieve bone marrow dysfunction. These results indicate that NPY promotes neuroprotection and restores bone marrow dysfunction from chemotherapy‐induced SNS injury through the Y1 receptor in macrophages. They also reveal a new role of NPY as a regulator of the bone marrow microenvironment and highlight the potential therapeutic value of this neuropeptide.     

Reduced EBF expression underlies loss of B‐cell potential of hematopoietic progenitors with age

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age‐related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2–5‐fold, while myeloid potential remains unchanged. This age‐related decrease in B‐cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B‐lineage specifying factors, EBF and Pax5. Notably, retrovirus‐mediated expression of EBF complemented the age‐related loss of B‐cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B‐cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B‐cell fate, without altering their potential to generate myeloid cells.     

Age-dependent macrophage functions in New Zealand black mice.

Various functions of macrophage derived from young (2-month-old) and old (14- to 17-month-old) New Zealand Black (NZB) mice with autoimmune disease were studied and compared with macrophage functions of age-matched BALB/c mice. Macrophages from young and old NZB mice demonstrated elevated levels of β-glucuronidase, cathepsin D, lysozyme, and DNase compared with those from age-matched BALB/c. DNase activity in the macrophages of NZB mice significantly increased with age. Macrophages from young and old NZB mice had greater phagocytic capacity for both ¹²⁵I-labeled Shigella flexneri and Staphylococcus albus than did BALB/c macrophages. NZB macrophages from both young and old mice had higher bactericidal activity against S. albus than those from age-matched BALB/c mice. The number of macrophage/granulocyte colony-forming cells (CFC) in both bone marrow and spleen was markedly higher in young and old NZB mice than in BALB/c mice. Colony-stimulating factor (CSF) released by macrophages derived from NZB mice had higher CFC activity than that released from macrophages of age-matched BALB/c mice. In NZB mice, the CSF activity significantly increased with age. It is suggested that potentiation of macrophage number and activity compensates for the deficiency of T cell functions in NZB mice with autoimmune disease.     

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer’s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently gene expression was evaluated.