Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non‐obese diabetic mice

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of β cells and perforin deficiency effectively reduces diabetes in non‐obese diabetic (NOD) mice, it can be deduced that β cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non‐obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB^–/– mice developed diabetes spontaneously with kinetics similar to those of wild‐type NOD (wt‐NOD) mice. Adoptive transfer study with regulatory T cell (T_reg)‐depleted splenocytes (SPCs) into NOD‐SCID mice or in‐vivo T_reg depletion by anti‐CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB^–/– mice and wt‐NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre‐diabetic NOD.GzmB^–/– mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide‐promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4⁺, CD8⁺ and CD4⁺CD25⁺ T cells in SPCs from NOD.GzmB^–/– mice than those from wt‐NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for β cell destruction in NOD mice.     

Induction of Bacterial Antigen-Specific Colitis by a Simplified Human Microbiota Consortium in Gnotobiotic Interleukin-10?/? Mice

We evaluated whether a simplified human microbiota consortium (SIHUMI) induces colitis in germfree (GF) 129S6/SvEv (129) and C57BL/6 (B6) interleukin-10-deficient (IL-10^−/−) mice, determined mouse strain effects on colitis and the microbiota, examined the effects of inflammation on relative bacterial composition, and identified immunodominant bacterial species in “humanized” IL-10^−/− mice. GF wild-type (WT) and IL-10^−/− 129 and B6 mice were colonized with 7 human-derived inflammatory bowel disease (IBD)-related intestinal bacteria and maintained under gnotobiotic conditions. Quantification of bacteria in feces, ileal and colonic contents, and tissues was performed using 16S rRNA gene selective quantitative PCR. Colonic segments were scored histologically, and gamma interferon (IFN-γ), IL-12p40, and IL-17 levels were measured in supernatants of unstimulated colonic tissue explants and of mesenteric lymph node (MLN) cells stimulated by lysates of individual or aggregate bacterial strains. Relative bacterial species abundances changed over time and differed between 129 and B6 mice, WT and IL-10^−/− mice, luminal and mucosal samples, and ileal and colonic or fecal samples. SIHUMI induced colitis in all IL-10^−/− mice, with more aggressive colitis and MLN cell activation in 129 mice. Escherichia coli LF82 and Ruminococcus gnavus lysates induced dominant effector ex vivo MLN TH1 and TH17 responses, although the bacterial mucosal concentrations were low. In summary, this study shows that a simplified human bacterial consortium induces colitis in ex-GF 129 and B6 IL-10^−/− mice. Relative concentrations of individual SIHUMI species are determined by host genotype, the presence of inflammation, and anatomical location. A subset of IBD-relevant human enteric bacterial species preferentially stimulates bacterial antigen-specific TH1 and TH17 immune responses in this model, independent of luminal and mucosal bacterial concentrations.     

Ambiguous Role of Interleukin-12 in Yersinia enterocolitica Infection in Susceptible and Resistant Mouse Strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4⁺ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR^−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55^−/−) mice. IFN-γR^−/− mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-γR^+/+ mice. Administration of IL-12 was protective in IFN-γR^+/+ mice but not in IFN-γR^−/− mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55^−/− mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

Regulatory Actions of Toll-Like Receptor 2 (TLR2) and TLR4 in Leishmania donovani Infection in the Liver

In livers of susceptible but self-curing C57BL/6 mice, intracellular Leishmania donovani infection enhanced Toll-like receptor 4 (TLR4) and TLR2 gene expression. In the liver, infected TLR4^−/− mice showed reduced gamma interferon (IFN-γ), tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS) mRNA expression, higher-level and slowly resolving infection, delayed granuloma formation, and little response to low-dose chemotherapy; in serum, the ratio of IFN-γ to interleukin 10 (IL-10) activity was decreased by 50%. In contrast, in TLR2^−/− mice, control of liver infection, parasite killing, and granuloma assembly were accelerated and chemotherapy''s efficacy enhanced. In livers of infected TLR2^−/− mice, mRNA expression was not increased for inflammatory cytokines or iNOS or decreased for IL-10; however, the serum IFN-γ/IL-10 ratio was increased 6.5-fold and minimal responses to IL-10 receptor blockade suggested downregulated IL-10. In established infection in wild-type mice, blockading TLR2 induced parasite killing and triggering TLR4 strengthened resistance and promoted chemotherapy''s effect. Thus, in experimental L. donovani infection in the liver, TLR4 signaling upregulates and TLR2 signaling downregulates macrophage antileishmanial activity, making both receptors potential therapeutic targets in visceral leishmaniasis for engagement (TLR4) or blockade (TLR2).     

Histopathological analysis of initial cellular response in TLR‐2 deficient mice experimentally infected by Leishmania (L.) amazonensis

Tegumentary leishmaniasis is an important public health problem in several countries. The capacity of the Leishmania species, at the initial moments of the infection, to invade and survive inside the host cells involves the interaction of surface molecules that are crucial in determining the evolution of the disease. Using C57BL/6 wild-type and TLR-2^−/− mice infected with L. (L.) amazonensis, we demonstrated that TLR-2^−/− mice presented eosinophilic granuloma in the ear dermis, different from C57BL/6 wild-type mice that presented a cellular profile characterized mainly by mononuclear cell infiltrates, besides neutrophils and eosinophils, during the two first week of infection. When the parasite load was evaluated, we found that the absence of TLR-2 lead to a significant reduction of the infection in deficient mice, when compared with C57BL/6 mice which were more susceptible to the infection. Using TLR-2 deficient mice, it was possible to show that the absence of this receptor determined the reduction of the parasite load and the recruitment of inflammatory cells during the two first weeks after L. (L.) amazonensis infection.     

Toll-Like Receptor 9 Signaling in Dendritic Cells Regulates Neutrophil Recruitment to Inflammatory Foci following Leishmania infantum Infection

Leishmania infantum is a protozoan parasite that causes visceral leishmaniasis (VL). This infection triggers dendritic cell (DC) activation through the recognition of microbial products by Toll-like receptors (TLRs). Among the TLRs, TLR9 is required for DC activation by different Leishmania species. We demonstrated that TLR9 is upregulated in vitro and in vivo during infection. We show that C57BL/6 mice deficient in TLR9 expression (TLR9^−/− mice) are more susceptible to infection and display higher parasite numbers in the spleen and liver. The increased susceptibility of TLR9^−/− mice was due to the impaired recruitment of neutrophils to the infection foci associated with reduced levels of neutrophil chemoattractants released by DCs in the target organs. Moreover, both Th1 and Th17 cells were also committed in TLR9^−/− mice. TLR9-dependent neutrophil recruitment is mediated via the MyD88 signaling pathway but is TIR domain-containing adapter-inducing interferon beta (TRIF) independent. Furthermore, L. infantum failed to activate both plasmacytoid and myeloid DCs from TLR9^−/− mice, which presented reduced surface costimulatory molecule expression and chemokine release. Interestingly, neutrophil chemotaxis was affected both in vitro and in vivo when DCs were derived from TLR9^−/− mice. Our results suggest that TLR9 plays a critical role in neutrophil recruitment during the protective response against L. infantum infection that could be associated with DC activation.     

Increase of tuberculous infection in the organs of B cell-deficient mice

Protective immunity against infection with Mycobacterium tuberculosis is imparted by T cells rather than antibodies, but B cells can play a role as antigen-presenting cells and in granuloma formation. We re-evaluated the role of B cells in the course of tuberculous infection in μ-chain knock-out (Ig⁻) mice. Surprisingly, the organs of M. tuberculosis-infected Ig⁻ mice were found to have three-to eight-fold elevated counts of viable bacilli compared with normal littermates at 3–6 weeks post-infection. Splenic interferon-gamma responses to whole antigen were unimpaired, whilst proliferation to certain mycobacterial peptides was found to be diminished. However, bacille Calmette–Guérin (BCG) vaccination significantly reduced the infection in Ig⁻ mice. The mechanisms by which B cells can influence primary tuberculous infection need further study.     

Human and mouse granzyme A induce a proinflammatory cytokine response

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.     

Genetic regulation of pristane‐induced oil granuloma responses

Oil granuloma (OG) induced by intraperitoneal injection of pristane represents a non-infectious granuloma. Oil granuloma has been characterized, but the regulation of its formation still remains unknown. To address this, we injected pristane into various mice deficient for genes including, linker for activation of T cells (LAT), μMT, LTα, TNFα, IL-6. T cell deficient mice (LAT^−/−) responded to pristane by developing serosal granuloma and mesenteric granuloma (MG) as in wild type mice. The absence of B cells blocked serosal granuloma (SG) formation and diminished MG development in response to pristane. However, even when a comparable number of B cells were present in the mesentery, the absence of TNFα resulted in similar defects in OG formation after pristane treatment, demonstrating that both B cells and TNFα are very crucial for pristane-induced OG formation. Interestingly, IL-6^−/− mice had intact MG formation; however, SG organization was impaired. These studies provide insight into granulomateous pathology induced by non-infectious substances for example, biomedical sutures.     

Recruitment of macrophages and polymorphonuclear leukocytes in L:yme carditis

Lyme arthritis, caused by the spirochete Borrelia burgdorferi, can be recurrent or prolonged, whereas Lyme carditis is mostly nonrecurring. A prominent difference between arthritis and carditis is the differential representation of phagocytes in these lesions: polymorphonuclear leukocytes (PMN) are more prevalent in the joint, and macrophages predominate in the heart lesion. We have previously shown differential efficiency of B. burgdorferi clearance by PMN and macrophages, and we now investigate whether these functional differences at the cellular level may contribute to the observed differences in organ-specific pathogenesis. When we infected mice lacking the neutrophil chemokine receptor (CXCR2^−/− mice) with spirochetes, we detected fewer PMN in joints and less-severe arthritis. Here we have investigated the effects of the absence of the macrophage chemokine receptor CCR2 on the development and resolution of Lyme carditis in resistant (C57BL/6J [B6]) and sensitive (C3H/HeJ [C3H]) strains of mice. In B6 CCR2^−/− mice, although inflammation in hearts is mild, we detected an increased burden of B. burgdorferi compared to that in wild-type (WT) mice, suggesting reduced clearance in the absence of macrophages. In contrast, C3H CCR2^−/− mice have severe inflammation but a decreased B. burgdorferi burden compared to that in WT C3H mice both at peak disease and during resolution. Histopathologic examination of infected hearts revealed that infected C3H CCR2^−/− animals have an increased presence of PMN, suggesting compensatory mechanisms of B. burgdorferi clearance in the hearts of infected C3H CCR2^−/− mice. The more efficient clearance of B. burgdorferi from hearts by CCR2^−/− versus WT C3H mice suggests a natural defect in the recruitment or function of macrophages in C3H mice, which may contribute to the sensitivity of this strain to B. burgdorferi infection.     

The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3^−/− mice had a higher mortality rate than C3^+/+ mice. Factor B^−/− mice, but not C4^−/− mice, infected with P. aeruginosa had a mortality rate similar to that of C3^−/− mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3^−/− mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3^+/+ mice at 24 h postinfection. In vitro, phagocytic cells from C3^+/+ or C3^−/− mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3^−/− mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1β (IL-1β), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3^+/+ mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.     

Critical Roles of Lysosomal Acid Lipase in Myelopoiesis

Lysosomal acid lipase (LAL) is a key enzyme that cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Genetic ablation of the lal gene (lal^−/−) in mice has resulted in a systemic increase of macrophages and neutrophils, causing severe inflammation and pathogenesis in multiple organs. We hypothesized that aberrant growth and differentiation of myeloid cells in lal^−/− mice arises from dysregulated production of progenitor cells in the bone marrow. Indeed, lal^−/− mice displayed increased numbers of primitive lin⁻Sca-1⁺c-Kit⁺ (LSK) cells and granulocyte-macrophage precursors (GMP). Increased high proliferative potential colony-forming cells (HPP-CFC) were enumerated from cultured lal^−/− bone marrow cells, as were significantly more CFU-GM, CFU-G, and CFU-M colonies. As a consequence, lal^−/− mice developed significant myeloid infiltration, particularly with CD11b⁺/Gr-1⁺ myeloid-derived suppressive cells in multiple organs. Both decreased apoptosis and increased proliferation contribute to the systemic increase of myeloid cells in lal^−/− myeloid cells. These lal^−/− CD11b⁺/Gr-1⁺ cells displayed suppressive activity on T cell proliferation and function in vitro. Bone marrow chimeras confirmed that the myeloproliferative disorder in lal^−/− mice was primarily attributable to autonomous defects in myeloid progenitor cells, although the hematopoietic microenvironment in the lal^−/− mice did not support hematopoiesis normally. These results provide evidence that LAL is an important regulator of myelopoiesis during hematopoietic development, differentiation, and homeostasis.Hematopoietic stem cells possess the capacity for self-renewal and the ability to differentiate into all lineages of mature hematopoietic cells.¹ The production of mature blood cells requires the sequential proliferation and differentiation of hematopoietic stem cells through a successive series of increasingly lineage-restricted intermediate progenitors including common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), and the megakaryocyte-erythroid progenitors (MEPs).² Because bone marrow-derived myeloid cells and lymphoid cells are effectors of inflammation and tissue remodeling in many organs, elucidating the molecular mechanisms governing hematopoiesis during the process of lineage commitment and lineage-specific expansion is critical for understanding inflammation-induced tissue damage and disease formation.Lysosomal acid lipase (LAL) is a lysosomal hydrolase. It hydrolyzes cholesteryl esters and triglycerides in the lysosome of cells to generate free cholesterol and free fatty acids. Ablating LAL (lal^−/−) in mice led to severe pathogenic phenotypes in multiple organs, including the adult liver, lung, spleen, adrenal glands, and small intestine.^3,4,5 These pathogenic changes in lal^−/− mice were highly associated with infiltration of macrophages and neutrophils,^3,6,7 suggesting that LAL plays a critical role in regulating the development, differentiation, and/or proliferation of myeloid lineage cells. This was supported by an observation that genetic intercross of a conditional transgenic mouse model in which LAL was specifically expressed in myeloid lineage cells with lal^−/− mice led to amelioration of the systemic inflammation and tissue pathogenesis.⁶To fully understand the dysregulated production of myeloid cells that are involved in lal^−/− disease formation, it is essential to elucidate the role of LAL in hematopoiesis. Our present results have revealed that LAL deficiency causes hematopoietic cell–intrinsic defects leading to profound blood phenotypes, especially an enhancement in CD11b⁺/Gr-1⁺ myeloid cells. LAL deletion in mice altered the frequency and the number of primitive IL7R⁻Lin⁻Sca-1⁺c-Kit⁺ (LSK), CMP, GMP, and MEP populations in the bone marrow and changed myeloid cell distribution in the bone marrow, peripheral blood, spleen, and distal organs (eg, lung). When LAL deficient hematopoietic progenitor cells were assessed in clonogenic assays, significant increases in the numbers of myeloid colony forming cells (CFCs) were enumerated. Both hematopoietic progenitor cell defects and a microenvironmental change in lal^−/− mice contributed to the above phenotype in bone marrow transplantation studies. These results demonstrate that neutral lipids and lipid mediators play important roles in normal myelopoiesis and contribute to abnormal inflammatory responses and disease formation in lal^−/− mice.     

Liver Xeno-Repopulation with Human Hepatocytes in Fah?/?Rag2?/? Mice after Pharmacological Immunosuppression

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury–induced donor hepatocyte selection: the uPA/SCID mice and Fah^−/− Rag2^−/−Il2rg^−/− mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah^−/−Rag2^−/− mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah^−/−Rag2^−/− mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.Human hepatocytes xeno-engrafted into the liver of immunodeficient mice could be used as a model to study human hepatitis virus infection in vivo as well as the efficacy of potential vaccines.^1,2,3,4,5,6 Engrafted human hepatocytes can be serially transplanted from primary mice into secondary mice without losing hepatic function.⁷ Mouse recipients of human liver cells must have two capabilities: robust liver repopulation and immune tolerance for human hepatocytes. Liver xeno-repopulation from human hepatocytes was first reported in uPA/Rag2^−/− mice¹ and uPA/SCID mice.^2,3,8 The levels of liver xeno-repopulation varies in several reports, ranging from 10% to as high as 90%.^1,8 Humanized livers in uPA/SCID mice are susceptible to hepatitis B virus (HBV)^1,2 and HCV^3,4 infection. However, uPA mice have several disadvantages: i) neonatal death during colony breeding; ii) transplantation of hepatocytes into newborn mice (within the second week of life) is technically difficult due to a bleeding disorder in the mice; iii) there is uncontrollable selection for donor cells; iv) there is autoreversion of endogenous hepatocytes; and v) kidney damage is induced by the human complement system.^1,2,8,9Recently, robust liver xeno-repopulation from human hepatocytes was found in Fah^−/−Rag2^−/−Il2rg^−/− mice, cross-bred from Fah^−/− mice and Rag2^−/−Il2rg^−/− mice.⁷ Fah^−/−Rag2^−/−Il2rg^−/− mice have advantages over previous immunodeficient uPA models.⁷ First, Rag2^−/−Il2rg^−/− mice lack B, T, and NK cells, rendering more complete immunodeficiency compared with either Rag2^−/− or SCID mice.¹⁰ Second, liver injury in Fah^−/− mice is controllable by switching on and off 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1, 3 cyclohexanedione (NTBC) administration.¹¹ NTBC inhibits accumulation of toxic metabolites in hepatocytes to maintain Fah^−/− mice in a healthy state. When the NTBC is removed, a powerful selection for fumaryl acetoacetate hydrolase (Fah) expressing cells is induced in the liver.¹² However, maintenance of Fah^−/−Rag2^−/−Il2rg^−/− mice during colony breeding, animal growing, and cell transplantation surgery are with high mortality in our experiments. The genotyping of animal offspring is overly elaborate. These concerns have not been discussed in previous publications^7,13 and may present a barrier to larger scale research projects.In comparison, Fah^−/−Rag2^−/− mice were much more tolerant of breeding and surgery procedures. However, Fah^−/−Rag2^−/− mice were thought to have no capacity for liver xeno-repopulation, because their NK cells are intact.⁷ We hypothesized that treatment of Fah^−/− Rag2^−/− mice with anti-asialo GM1 could result in complete depletion of NK cells as seen in Fah^−/−Rag2^−/− Il2rg^−/− mice.^14,15 We further tested the combined treatments of both anti-asialo GM1 and the immunosuppressor tacrolimus (FK506) to Fah^−/−Rag2^−/− mice.^16,17 The results indicated that the combined treatments enabled Fah^−/−Rag2^−/− recipients to have a high level of liver xeno-repopulation by human hepatocytes as seen in Fah^−/−Rag2^−/−Il2rg^−/− mice. Our results revealed a new and easily controlled mouse model with humanized liver. Using the same treatments, liver xeno-repopulation with human fetal liver progenitor cells was also achieved in Fah^−/−Rag2^−/− mice. Finally, for the first time, we were able to prove that human HBV actively replicated in the humanized Fah^−/−Rag2^−/− mice and that viral proteins were released in the serum of humanized Fah^−/−Rag2^−/− mice, which showed no significant difference with previous reports of human HBV infection in humanized uPA/SCID mice.^1,2,5     

CD14 Influences Host Immune Responses and Alternative Activation of Macrophages during Schistosoma mansoni Infection

Antigen-presenting cell (APC) plasticity is critical for controlling inflammation in metabolic diseases and infections. The roles that pattern recognition receptors (PRRs) play in regulating APC phenotypes are just now being defined. We evaluated the expression of PRRs on APCs in mice infected with the helminth parasite Schistosoma mansoni and observed an upregulation of CD14 expression on macrophages. Schistosome-infected Cd14^−/− mice showed significantly increased alternative activation of (M2) macrophages in the livers compared to infected wild-type (wt) mice. In addition, splenocytes from infected Cd14^−/− mice exhibited increased production of CD4⁺-specific interleukin-4 (IL-4), IL-5, and IL-13 and CD4⁺Foxp3⁺IL-10⁺ regulatory T cells compared to cells from infected wt mice. S. mansoni-infected Cd14^−/− mice also presented with smaller liver egg granulomas associated with increased collagen deposition compared to granulomas in infected wt mice. The highest expression of CD14 was found on liver macrophages in infected mice. To determine if the Cd14^−/− phenotype was in part due to increased M2 macrophages, we adoptively transferred wt macrophages into Cd14^−/− mice and normalized the M2 and CD4⁺ Th cell balance close to that observed in infected wt mice. Finally, we demonstrated that CD14 regulates STAT6 activation, as Cd14^−/− mice had increased STAT6 activation in vivo, suggesting that lack of CD14 impacts the IL-4Rα-STAT6 pathway, altering macrophage polarization during parasite infection. Collectively, these data identify a previously unrecognized role for CD14 in regulating macrophage plasticity and CD4⁺ T cell biasing during helminth infection.     

Influence of Acute-Phase Parasite Load on Pathology, Parasitism, and Activation of the Immune System at the Late Chronic Phase of Chagas’ Disease

To obtain low and high parasite loads in the acute phase of Chagas’ disease, A/J mice were infected with 10³ or 10⁵ Trypanosoma cruzi trypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8⁺ and B220⁻ CD5⁻ cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220⁺), CD4⁺, and CD8⁺ large lymphocytes; (iii) a shift of CD4⁺ cells towards a CD45RB^Low phenotype; (iv) increased frequencies of both CD45RB^Low and CD45RB^High large CD4⁺ cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase of T. cruzi infection influences the activation of the immune system and development of Chagas’ disease pathology at the late chronic phase of the disease.     

L-Plastin Is Essential for Alveolar Macrophage Production and Control of Pulmonary Pneumococcal Infection

We report that mice deficient for the hematopoietic-specific, actin-bundling protein L-plastin (LPL) succumb rapidly to intratracheal pneumococcal infection. The increased susceptibility of LPL^−/− mice to pulmonary pneumococcal challenge correlated with reduced numbers of alveolar macrophages, consistent with a critical role for this cell type in the immediate response to pneumococcal infection. LPL^−/− mice demonstrated a very early clearance defect, with an almost 10-fold-higher bacterial burden in the bronchoalveolar lavage fluid 3 h following infection. Clearance of pneumococci from the alveolar space in LPL^−/− mice was defective compared to that in Rag1^−/− mice, which lack all B and T lymphocytes, indicating that innate immunity is defective in LPL^−/− mice. We did not identify defects in neutrophil or monocyte recruitment or in the production of inflammatory cytokines or chemokines that would explain the early clearance defect. However, efficient alveolar macrophage regeneration following irradiation required LPL. We thus identify LPL as being key to alveolar macrophage development and essential to an effective antipneumococcal response. Further analysis of LPL^−/− mice will illuminate critical regulators of the generation of alveolar macrophages and, thus, effective pulmonary innate immunity.     

Importance of T cells, gamma interferon, and tumor necrosis factor in immune control of the rapid grower Mycobacterium abscessus in C57BL/6 mice

Mycobacterium abscessus is an emerging rapidly growing mycobacterium that causes tuberculous-like lesions in humans. We studied the immune control of this organism in C57BL/6 mice challenged intravenously with 10⁷ CFU. Bacteria were eliminated from both the spleen and the liver within 90 days, and liver histology showed organized granulomatous lesions. A T- and B-cell requirement was investigated by challenging Rag2^−/−, Cd3^−/−, and μMT^−/− mice. Rag2^−/− and Cd3^−/− mice were significantly impaired in the ability to clear M. abscessus from the liver and spleen, and μMT^−/− mice were significantly impaired in the ability to clear M. abscessus from the liver, suggesting that infection control was primarily T cell dependent in the spleen and both T and B cell dependent in the liver. The liver granulomatous response was similar to that of wild-type controls in μMT^−/− mice but completely absent in Cd3^−/− and Rag2^−/− mice. We studied the involvement of gamma interferon (IFN-γ) and tumor necrosis factor (TNF) by challenging C57BL/6 mice deficient in the IFN-γ receptor (Ifngr1^−/−) and in TNF (Tnf^−/−). Ifngr1^−/− mice were significantly impaired in M. abscessus control both in the spleen and in the liver, and granulomas were profoundly altered. The effect was even more substantial in Tnf^−/− mice; they failed to control M. abscessus infection in the liver and died within 20 to 25 days after infection with many hepatic inflammatory foci and major lesions of ischemic necrosis in the liver and kidney. These features were not observed with the closely related species M. chelonae. T-cell immunity, IFN-γ, and TNF are central factors for the control of M. abscessus in C57BL/6 mice, as they are for the control of pathogenic slowly growing mycobacteria.     

Activated Pulmonary Macrophages Are Insufficient for Resistance against Pneumocystis carinii

CD4⁺ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (Aβ^−/−), T-cell receptor αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-γ) receptor (IFN-γ-R^−/−) or tumor necrosis factor alpha (TNF-α) type I receptor (p55) (TNF-α-RI^−/−) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-γ, TNF-α, and IL-12 in diseased TCRβ^−/− and RAG-1^−/− mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid Aβ^−/− mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-γ nor TNF-α appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4⁺ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4⁺ TCRαβ cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4⁺ T lymphocytes, must contribute to successful resistance against P. carinii.Immunocompromised patients, especially those suffering from AIDS, are at elevated risk of acquiring Pneumocystis carinii pneumonia (PCP), a major cause of premature mortality among AIDS patients (8, 35, 53). Various studies have emphasized that CD4⁺ T lymphocytes play a pivotal role in the orchestration of resistance to P. carinii (22, 43, 45), an opportunistic fungus, but the mechanisms underlying protection remain a conundrum. Pulmonary macrophages are considered the main effector cells in clearing the immunocompetent host from invading P. carinii organisms (25). It seems conceivable, therefore, that macrophage-activating functions mediated by CD4⁺ T cells are central to resistance. Impaired gamma interferon (IFN-γ) production by T cells from AIDS patients is thought to enhance susceptibility to P. carinii (34, 41). This notion is supported by reports that application of exogenous IFN-γ ameliorates disease in experimental animal models (2, 45). In contrast, in vivo neutralization of IFN-γ in spleen cell-reconstituted severe combined immunodeficiency (SCID) mice by a specific monoclonal antibody (MAb) does not affect parasite clearance (5). Further studies point to a critical role of tumor necrosis factor alpha (TNF-α) (5) and interleukin-1 (IL-1) (6) in maintaining an immunocompetent state. Both cytokines are mainly produced by macrophages and induce inflammatory responses (4, 10, 26). Overall, these findings support involvement of macrophage-derived cytokines in successful host resistance against P. carinii.To analyze in more depth the role of inflammatory and Th1/Th2-related pulmonary defense mechanisms in control of aerogenically acquired PCP, we took advantage of naturally susceptible gene disruption mutant mice lacking major histocompatibility complex (MHC) class II molecules (and therefore conventional CD4⁺ T cells) (Aβ^−/−), T-cell receptor (TCR) αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) (19). We further exploited mice deficient in the IFN-γ receptor (IFN-γ-R^−/−) or the TNF-α type I receptor (p55) (TNF-α-RI^−/−) to analyze their capacity to cope with aerogenic P. carinii organisms.Bronchoalveolar lavage (BAL) cells of healthy and diseased mice were investigated for expression of the proinflammatory cytokines IL-1, TNF-α, IFN-γ, and IL-12, as well as IL-4, IL-5, and IL-10. The latter three cytokines counteract IFN-γ- and IL-12-mediated responses but participate in protection against certain extracellular pathogens (9). Moreover, production of superoxide (SO) and nitric oxide (NO), putative effector molecules of antimicrobial defense, was taken as a further indicator of macrophage activation. Contact with foreign material induces a rapid respiratory burst in professional phagocytes which results in SO production as a first line of defense. SO has been implicated in destruction of P. carinii (31), whereas NO produced by IFN-γ-stimulated macrophages encountering pathogens (4, 18, 30) does not appear to participate in control of P. carinii infection (47). Of further interest was the role of macrophage-expressed mannose receptors (MR) and scavenger receptors (SR). MR were previously found crucial for mediating P. carinii internalization (11, 37). The relevance of SR with respect to PCP has not been evaluated, but they are mainly expressed by tissue macrophages (36) and nonspecifically bind a large array of molecules, including surface molecules of microorganisms (39). Receptors with such broad pattern reactivity may be involved in direct differentiation of self from non-self, and recent data suggest that not only MR but also SR aid pattern recognition by macrophages and subsequent internalization of invading pathogens (27).We found that BAL cells from P. carinii-diseased RAG-1^−/− and TCRβ^−/− mutants secreted elevated IFN-γ, TNF-α, IL-12, NO, and SO levels and expressed IL-1 mRNA. In contrast, cells from morbid Aβ^−/− mice produced IL-1 mRNA and high levels of NO only, whereas all other parameters were low to absent in these mutants. SR were expressed on pulmonary macrophages of all diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants, whereas MR were also expressed by macrophages of healthy animals. Yet, the apparently activated phagocyte system in the lung, most pronounced in morbid TCRβ^−/− and RAG-1^−/− mutant mice, was insufficient for protection against natural P. carinii infection. Elevated levels of IFN-γ and TNF-α in morbid mutants (not in Aβ^−/− mice) and the naturally resistant status of IFN-γ-R^−/− and TNF-α-RI^−/− mice further argue not only for independence from IFN-γ and TNF-α. Our findings indicate that CD4⁺ αβ T lymphocytes prevent and clear infection with P. carinii by mechanisms distinct from, or in addition to, pulmonary macrophage activation.(This study is part of the Ph.D. thesis of R. Hanano.)     

Neither Dectin-2 nor the Mannose Receptor Is Required for Resistance to Coccidioides immitis in Mice

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2^−/− and MR^−/− myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.     

The in vivo induction of lymphocyte apoptosis in MRL-lpr/lpr mice treated with FTY720

The in vitro treatment of lpr thymocytes with FTY720 resulted in a dose-dependent reduction in cell viability due to apoptosis. In order to study the effect of FTY720 in vivo, lpr mice received an oral daily dose of 1 mg/kg FTY720 for 14 days, beginning at 16 weeks of age which was when the animals were developing massive lymphoadenopathy. Compared with untreated lpr mice, FTY720-treated lpr mice had significantly prolonged lives. At 24 weeks of age, treated mice demonstrated markedly reduced weights of the spleen and lymph nodes, and the proportion of CD3⁺B220⁺ and CD4⁻CD8⁻ cells in the thymus, spleen and lymph nodes decreased markedly. In addition, in these mice the percentage of CD4⁺CD8⁺ and CD3⁻B220⁻ cells in the thymus and the percentage of CD4⁺CD8⁻, CD4⁻CD8⁺, CD3⁺B220⁻ and CD3⁻B220⁺ cells in the spleen returned to almost the normal values observed in wild-type mice. Histological observation 1 day after the final administration of FTY720 revealed a remarkable infiltration of neutrophils in the lymphoid organs. Apoptotic cells were detected in all the lymphoid organs using in situDNA nick-end labelling. Electron microscopy showed that the apoptotic cells were ingested by phagocytes. FTY720 therapy is thus highly effective in Fas-mutant animals with abnormally expanding lymphocytes.