Sensing of Interleukin-1 Cytokines during Streptococcus pneumoniae Colonization Contributes to Macrophage Recruitment and Bacterial Clearance

Streptococcus pneumoniae (the pneumococcus), a leading cause of bacterial disease, is most commonly carried in the human nasopharynx. Colonization induces inflammation that promotes the organism''s growth and transmission. This inflammatory response is dependent on intracellular sensing of bacterial components that access the cytosolic compartment via the pneumococcal pore-forming toxin pneumolysin. In vitro, cytosolic access results in cell death that includes release of the proinflammatory cytokine interleukin-1β (IL-1β). IL-1 family cytokines, including IL-1β, are secreted upon activation of inflammasomes, although the role of this activation in the host immune response to pneumococcal carriage is unknown. Using a murine model of pneumococcal nasopharyngeal colonization, we show that mice deficient in the interleukin-1 receptor type 1 (Il1r1^−/−) have reduced numbers of neutrophils early after infection, fewer macrophages later in carriage, and prolonged bacterial colonization. Moreover, intranasal administration of Il-1β promoted clearance. Macrophages are the effectors of clearance, and characterization of macrophage chemokines in colonized mice revealed that Il1r1^−/− mice have lower expression of the C-C motif chemokine ligand 6 (CCL6), correlating with reduced macrophage recruitment to the nasopharynx. IL-1 family cytokines are known to promote adaptive immunity; however, we observed no difference in the development of humoral or cellular immunity to pneumococcal colonization between wild-type and Il1r1^−/− mice. Our findings show that sensing of IL-1 cytokines during colonization promotes inflammation without immunity, which may ultimately benefit the pneumococcus.     

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.     

MyD88-Dependent Signaling Drives Host Survival and Early Cytokine Production during Histoplasma capsulatum Infection

The ability of the innate immune system to trigger an adaptive T cell response is critical to resolution of infection with the fungal pathogen Histoplasma capsulatum. However, the signaling pathways and cell types involved in the recognition of and response to this respiratory pathogen remain poorly defined. Here, we show that MyD88, an adaptor protein vital to multiple innate immune pathways, is critically required for the host response to Histoplasma. MyD88-deficient (MyD88^−/−) mice are unable to control the fungal burden and are more sensitive to Histoplasma infection than wild-type, Dectin-1^−/−, or interleukin 1 receptor-deficient (IL-1R^−/−) mice. We found that MyD88 is necessary for the production of key early inflammatory cytokines and the subsequent recruitment of inflammatory monocytes to the lung. In both our in vitro and ex vivo analyses, MyD88 was intrinsically required in dendritic cells and alveolar macrophages for initial cytokine production. Additionally, MyD88-deficient bone marrow-derived dendritic cells fail to efficiently control fungal growth when cocultured with primed splenic T cells. Surprisingly, mice that lack MyD88 only in dendritic cells and alveolar macrophages are competent for early cytokine production and normal survival, indicating the presence of compensatory and redundant MyD88 signaling in other cell types during infection. Ultimately, global MyD88 deficiency prevents proper T cell activation and gamma interferon (IFN-γ) production, which are critical for infection resolution. Collectively, this work reveals a central role for MyD88 in coordinating the innate and adaptive immune responses to infection with this ubiquitous fungal pathogen of humans.     

Analysis of murine genetic predisposition to pneumococcal infection reveals a critical role of alveolar macrophages in maintaining the sterility of the lower respiratory tract

The study of pathogenic mechanisms of disease can be greatly facilitated by studying genetic differences in susceptibility to infection. In the present study, we compared the severity of pneumococcal infection in C57BL/6 (B6) and 129Sv mice. The results showed that 129Sv mice were remarkably more susceptible to pneumococcal infection than B6 mice. Bacterial clearance, proinflammatory mediators, leukocyte recruitment, and phagocyte activities were measured to examine potential immune factors associated with differences in susceptibility to pneumococcal infection. The greater susceptibility of 129Sv mice was associated only with inadequate alveolar macrophage bacterial killing, as indicated by significantly decreased initial bacterial clearance from the respiratory tract. Effective pneumococcal clearance was not dependent upon Toll-like receptor 2 (TLR2) expression, oxidative stress, or matrix metallopeptidase 12 (MMP-12) expression. Furthermore, phagocytosis analysis suggested that the deficiency found in 129Sv alveolar macrophages was not due to a lack of bacterial recognition but, rather, to reduced bacterial uptake. In conclusion, our findings indicate a crucial role of alveolar macrophage phagocytosis during innate defense against pneumococcal infection, which may explain the association of host genetic risk factors with predisposition to pneumococcal infection.     

Toll-Like Receptor 2 (TLR2) and TLR9 Play Opposing Roles in Host Innate Immunity against Salmonella enterica Serovar Typhimurium Infection

Toll-like receptors (TLRs) are evolutionarily conserved host proteins that are essential for effective host defense against pathogens. However, recent studies suggest that some TLRs can negatively regulate immune responses. We observed here that TLR2 and TLR9 played opposite roles in regulating innate immunity against oral infection of Salmonella enterica serovar Typhimurium in mice. While TLR9^−/− mice exhibited shortened survival, an increased cytokine storm, and more severe Salmonella hepatitis than wild-type (WT) mice, TLR2^−/− mice exhibited the opposite phenomenon. Further studies demonstrated that TLR2 deficiency and TLR9 deficiency in macrophages both disrupted NK cell cytotoxicity against S. Typhimurium-infected macrophages by downregulating NK cell degranulation and gamma interferon (IFN-γ) production through decreased macrophage expression of the RAE-1 NKG2D ligand. But more importantly, we found that S. Typhimurium-infected TLR2^−/− macrophages upregulated inducible nitric oxide synthase (iNOS) expression, resulting in a lower bacterial load than that in WT macrophages in vitro and livers in vivo as well as low proinflammatory cytokine levels. In contrast, TLR9^−/− macrophages showed decreased reactive oxygen species (ROS) expression concomitant with a high bacterial load in the macrophages and in livers of TLR9^−/− mice. TLR9^−/− macrophages were also more susceptible than WT macrophages to S. Typhimurium-induced necroptosis in vitro, likely contributing to bacterial spread and transmission in vivo. Collectively, these findings indicate that TLR2 negatively regulates anti-S. Typhimurium immunity, whereas TLR9 is vital to host defense and survival against S. Typhimurium invasion. TLR2 antagonists or TLR9 agonists may thus serve as potential anti-S. Typhimurium therapeutic agents.     

Intrinsic T‐ and B‐cell defects impair T‐cell‐dependent antibody responses in mice lacking the actin‐bundling protein L‐plastin

Germinal center development, critical for long‐term humoral immunity, requires the trafficking of T and B lymphocytes to defined tissues and locations after antigenic challenge. The molecular mechanisms that support lymphocyte trafficking through the linkage of extracellular chemotactic and adhesive cues to the actin cytoskeleton are not yet fully defined. We have previously identified the actin‐bundling protein L‐plastin (LPL) as a requisite intermediary in both naive B and T lymphocyte migration and in T‐cell activation. We tested the hypothesis that humoral immunity would require LPL. We show that mice lacking LPL demonstrated defective germinal center formation and reduced production of T‐cell‐dependent antibodies. T cells from LPL^?/? mice exhibited defective expansion of the follicular helper T population. Reduced expansion of LPL^?/? follicular helper T cells correlated with impaired trafficking to or retention of cells in the spleen following challenge, highlighting the importance of initial lymphocyte recruitment to the eventual success of the immune response. Furthermore, LPL^?/? B cells demonstrated cell‐intrinsic defects in population expansion and in differentiation into germinal center B cells. LPL thus modulates both T‐ and B‐cell function during the germinal center reaction and the production of T‐cell‐dependent antibody responses.     

Role of Dectin-2 for Host Defense against Systemic Infection with Candida glabrata

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2^−/−) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2^−/− mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2^−/− macrophages and neutrophils. Neutrophils of Dectin-2^−/− mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.     

A serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibody requires Fcγ receptor III and macrophages to mediate protection against pneumococcal pneumonia in mice

Antibodies to pneumococcal capsular polysaccharide (PPS) are required for PPS-based vaccine-mediated protection against Streptococcus pneumoniae. Previous work established that 1E2, a mouse IgG1 to PPS3 that does not induce serotype 3 (ST3) S. pneumoniae killing by phagocytes in vitro, protects mice from death after intranasal infection with ST3, but its efficacy was abrogated in FcγR (F common gamma receptor)-deficient mice. In this study, we determined whether 1E2 efficacy against pulmonary ST3 infection requires FcγRIII. 1E2 did not protect FcγRIII-deficient (FcγRIII^−/−) mice. Studies of the mechanism of 1E2-mediated effects showed that it resulted in a marked reduction in lung inflammation in ST3-infected wild-type (Wt [C57BL/6]) mice that was abrogated in FcγRIII^−/− mice. 1E2 had no effect on early bacterial clearance in the lungs of ST3-infected Wt, FcγRIIB^−/−, or FcγRIII^−/− mice, but it reduced levels of bacteremia and serum macrophage inflammatory protein-2) (MIP-2), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in Wt and FcγRIIB^−/− mice, strains in which it is protective. As previous work showed that neutrophils were dispensable for 1E2 efficacy, we investigated whether macrophages are required for 1E2 efficacy against intranasal infection with ST3 and found that its efficacy was abrogated in Wt mice depleted of macrophages intranasally. In vitro studies revealed that1E2 promoted ST3 internalization by naïve alveolar macrophages but did not induce early intracellular killing. Macrophages from 1E2-treated ST3-infected mice studied ex vivo exhibited more apoptosis than those from FcγRIII^−/− mice. These findings suggest that 1E2 mediates protection against ST3 in mice by affecting the inflammatory response, perhaps in part via macrophage apoptosis, rather than by inducing early bacterial clearance.     

The Absence of Hck,Fgr, and Lyn Tyrosine Kinases Augments Lung Innate Immune Responses to Pneumocystis murina

Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1β (IL-1β), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn^−/− mice 3 days postchallenge, although the level was less than that observed in Hck^−/− Fgr^−/− Lyn^−/− mice. A correlate to augmented clearance of P. murina in Hck^−/− Fgr^−/− Lyn^−/− mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.Although the advent of prophylactic therapy against Pneumocystis jirovecii alone or in combination with highly active antiretroviral therapy has reduced the incidence of pneumonia caused by this fungal pathogen, this infection remains the most prevalent opportunistic infection in individuals with AIDS (25). Furthermore, individuals receiving immunosuppressive therapies, such as individuals undergoing solid organ or hematopoietic cell transplantation, are a growing population susceptible to Pneumocystis pneumonia (34) (33). These observations warrant a more thorough examination of interactions between Pneumocystis and the host in order to define and develop new vaccine and immunotherapeutic approaches to treat Pneumocystis pneumonia.An intense area of research over the last decade is investigating how lung immune cells recognize and respond to inhaled pathogens, such as P. murina. After inhalation of P. murina into the lungs, one of the first interactions with the host is recognition by the alveolar macrophage. Our laboratory has previously reported that alveolar macrophages recognize P. murina via the beta-glucan receptor dectin-1 (37). Recognition by dectin-1 leads to internalization of P. murina and subsequent killing of the organism, as well as elaboration of the neutrophil-attracting chemokine CXCL2/MIP-2 (37). Toll-like receptor 2 (TLR2) is an additional receptor expressed by alveolar macrophages that mediates CXCL2/MIP-2 production (54) and, in humans, interleukin-8 (IL-8) production in cooperation with the macrophage mannose receptor (39). P. murina infection in TLR2^−/− mice is prolonged and associated with a lack of inflammatory responsiveness (51). Other studies have implicated TLR4 in alveolar macrophage recognition of P. murina (6).Immunoreceptors expressed by cells of the innate immune system are abundant and fall into many different categories, such as scavenger receptors, integrins, immunoglobulin (Ig) superfamily receptors, C-type lectin receptors, and TLRs (41). As expected based on this diversity, immunoreceptor signaling in innate cells is a complex process that differs greatly for different types of receptors. For example, receptors in the C-type lectin family often utilize an immunotyrosine activation motif (ITAM) in the cytoplasmic tail for signaling (2). Receptors in the Ig superfamily, such as SIRPα and Siglec3, contain immunotyrosine inhibitory motifs (ITIMs) that initiate regulatory signals, whereas members of the TLR family signal through multiple intermediate proteins, such as TIRAP, TRIF, and MyD88 (28). One commonality in innate immunoreceptor signaling is the role of Src family tyrosine kinases (SFKs). Our current understanding of how SFKs function is due in large part to studies that have characterized ITAM-associated Fc receptor (FcγR) signaling (32). In FcγR signaling, SFKs phosphorylate two tyrosine residues in the ITAM domain, which leads to the recruitment of Syk and subsequent activation of cellular responses, such as phagocytosis and cytokine and chemokine production (32).An equally important function of SFKs is to phosphorylate ITIMs, which leads to the recruitment of SHP-1 or SHIP-1 phosphatases and subsequent regulation or inhibition of responses (21). The phosphorylation of ITIMs by SFKs is often responsible for the regulation of responses initiated by many types of immunoreceptors. Mice deficient in the SFKs Hck and Fgr have enhanced chemokine receptor signaling as a result of lower phosphorylation of the ITIM in the inhibitory receptor paired Ig receptor B (PIR-B) (55). Mice deficient in PIR-B were also found to be hyperresponsive to chemokine signaling (55). PIR-B ITIM activation has also been shown to regulate TLR-mediated macrophage responses to some gram-positive and gram-negative bacteria (26). Other studies have shown that the SFK Lyn phosphorylates an ITIM of platelet endothelial cell adhesion molecule 1 in mast cells, leading to regulated FcɛRI responses (45).Macrophage-mediated recognition of P. murina involves a variety of receptors that may be dependent on SFKs for induction as well as the regulation of responses; therefore, we sought to investigate the role of SFKs in innate immune responses to P. murina. In this study, we made the surprising observation that deficiency of Hck, Fgr, and Lyn resulted in paradoxically augmented lung clearance of P. murina and enhanced cytokine and chemokine production. This response was not due to impaired PIR-B inhibitory responses as PIR-B^−/− mice were not capable of enhanced P. murina lung clearance. We propose that the SFKs Hck, Fgr, and Lyn regulate innate immune responses to P. murina and that, therefore, novel therapeutics to control the activity of SFKs may be beneficial in treating pulmonary infections.     

Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4⁺ T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2^−/− mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2^−/− mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR^−/− nor RAG/IL-4Rα^−/− mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR^−/− mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα^−/− mice. RAG/IFN-γR^−/− mice had elevated numbers of lung CD4⁺ T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8⁺ T suppressor cells. Impaired lung CD8⁺ T cell responses in RAG/IFN-γR^−/− mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8⁺ T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.     

MyD88 signaling contributes to early pulmonary responses to Aspergillus fumigatus

Toll-like receptors and the β-glucan receptor, dectin-1, mediate macrophage inflammatory responses to Aspergillus fumigatus through MyD88-dependent and -independent signaling mechanisms; however, pulmonary inflammatory responses in MyD88-deficient mice challenged with A. fumigatus are poorly defined. The role of MyD88 signaling in early pulmonary inflammation and fungal clearance was evaluated in C57BL/6J wild-type (WT) and MyD88-deficient (MyD88^−/−) mice. Early (<48 h) after infection, MyD88^−/− mice had higher fungal burdens than those of WT mice, although fungal burdens rapidly declined (>72 h) in both. MyD88^−/− mice had less consolidated inflammation, with fewer NK cells, in lung tissue early (24 h) after infection than did WT mice. At the latter time point, MyD88^−/− mouse lungs were characterized by a large amount of necrotic cellular debris and fibrin, while WT lungs had organized inflammation. Although there were equivalent numbers of macrophages in WT and MyD88^−/− mouse lung tissues, MyD88^−/− cells demonstrated delayed uptake of green fluorescent protein-expressing A. fumigatus (GFP-Af293); histologically, MyD88^−/− mouse lungs had more hyphal invasion of terminal airways and vessels, the appearance of bronchiolar epithelial cell necrosis, and necrotizing vasculitis. MyD88^−/− lung homogenates contained comparatively decreased amounts of interleukin-1β (IL-1β), IL-6, KC, and gamma interferon and paradoxically increased amounts of tumor necrosis factor alpha and macrophage inflammatory protein 1α. These data indicate that the MyD88-dependent pathway mediates acute pulmonary fungal clearance, inflammation, and tissue injury very early after infection. Resolution of abnormalities within a 3-day window demonstrates the importance of redundant signaling pathways in mediating pulmonary inflammatory responses to fungi.     

Impaired Innate Immunity in Mice Deficient in Interleukin-1 Receptor-Associated Kinase 4 Leads to Defective Type 1 T Cell Responses,B Cell Expansion,and Enhanced Susceptibility to Infection with Toxoplasma gondii

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a member of the IRAK family and has an important role in inducing the production of inflammatory mediators. This kinase is downstream of MyD88, an adaptor protein essential for Toll-like receptor (TLR) function. We investigated the role of this kinase in IRAK4-deficient mice orally infected with the cystogenic ME49 strain of Toxoplasma gondii. IRAK4^−/− mice displayed higher morbidity, tissue parasitism, and accelerated mortality than the control mice. The lymphoid follicles and germinal centers from infected IRAK4^−/− mice were significantly smaller. We consistently found that IRAK4^−/− mice showed a defect in splenic B cell activation and expansion as well as diminished production of gamma interferon (IFN-γ) by T lymphocytes. The myeloid compartment was also affected. Both the frequency and ability of dendritic cells (DCs) and monocytes/macrophages to produce IL-12 were significantly decreased, and resistance to infection with Toxoplasma was rescued by treating IRAK4^−/− mice with recombinant IL-12 (rIL-12). Additionally, we report the association of IRAK4 haplotype-tagging single nucleotide polymorphisms (tag-SNPs) with congenital toxoplasmosis in infected individuals (rs1461567 and rs4251513, P < 0.023 and P < 0.045, respectively). Thus, signaling via IRAK4 is essential for the activation of innate immune cells, development of parasite-specific acquired immunity, and host resistance to infection with T. gondii.     

Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and Toll-like receptor 2 knockout mice and protects against reinfection

Coxiella burnetii is a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuated C. burnetii phase II (having a truncated “O” chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-γ^−/−), BALB/c, Toll-like receptor 2 knockout (TLR2^−/−), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-γ^−/− and TLR2^−/− mice, which showed elevated rectal temperatures that were sustained for ~15 days with transient increases in splenic weights. Reinfection of IFN-γ^−/− and TLR2^−/− mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2^−/− mouse spleens was observed, greater colonization was evident in the IFN-γ^−/− mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-γ^−/− macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2^−/− macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.     

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.     

MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection

Spotted fever group rickettsiae cause potentially life-threatening infections throughout the world. Several members of the Toll-like receptor (TLR) family are involved in host response to rickettsiae, and yet the mechanisms by which these TLRs mediate host immunity remain incompletely understood. In the present study, we found that host susceptibility of MyD88^−/− mice to infection with Rickettsia conorii or Rickettsia australis was significantly greater than in wild-type (WT) mice, in association with severely impaired bacterial clearance in vivo. R. australis-infected MyD88^−/− mice showed significantly lower expression levels of gamma interferon (IFN-γ), interleukin-6 (IL-6), and IL-1β, accompanied by significantly fewer inflammatory infiltrates of macrophages and neutrophils in infected tissues, than WT mice. The serum levels of IFN-γ, IL-12, IL-6, and granulocyte colony-stimulating factor were significantly reduced, while monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and RANTES were significantly increased in infected MyD88^−/− mice compared to WT mice. Strikingly, R. australis infection was incapable of promoting increased expression of MHC-II^high and production of IL-12p40 in MyD88^−/− bone marrow-derived dendritic cells (BMDCs) compared to WT BMDCs, although costimulatory molecules were upregulated in both types of BMDCs. Furthermore, the secretion levels of IL-1β by Rickettsia-infected BMDCs and in the sera of infected mice were significantly reduced in MyD88^−/− mice compared to WT controls, suggesting that in vitro and in vivo production of IL-1β is MyD88 dependent. Taken together, our results suggest that MyD88 signaling mediates instructive signals in DCs and secretion of IL-1β and type 1 immune cytokines, which may account for the protective inflammatory response during rickettsial infection.     

Vaccinated C57BL/6 Mice Develop Protective and Memory T Cell Responses to Coccidioides posadasii Infection in the Absence of Interleukin-10

High concentrations of lung tissue-associated interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, correlate with susceptibility of mice to Coccidioides spp. infection. In this study, we found that macrophages, dendritic cells, neutrophils, and both CD8⁺ and CD4⁺ T cells recruited to Coccidioides posadasii-infected lungs of nonvaccinated and vaccinated mice contributed to the production of IL-10. The major IL-10-producing leukocytes were CD8⁺ T cells, neutrophils, and macrophages in lungs of nonvaccinated mice, while both Foxp3⁺ and Foxp3⁻ subsets of IL-10⁺ CD4⁺ T cells were significantly elevated in vaccinated mice. Profiles of the recruited leukocytes in lungs revealed that only CD4⁺ T cells were significantly increased in IL-10^−/− knockout mice compared to their wild-type counterparts. Furthermore, ex vivo recall assays showed that CD4⁺ T cells isolated from vaccinated IL-10^−/− mice compared to vaccinated wild-type mice produced significantly higher amounts of IL-2, gamma interferon (IFN-γ), IL-4, IL-6, and IL-17A in the presence of a coccidioidal antigen, indicating that IL-10 suppresses Th1, Th2, and Th17 immunity to Coccidioides infection. Analysis of absolute numbers of CD44⁺ CD62L⁻ CD4⁺ T effector memory T cells (T_EM) and IFN-γ- and IL-17A-producing CD4⁺ T cells in the lungs of Coccidioides-infected mice correlated with better fungal clearance in nonvaccinated IL-10^−/− mice than in nonvaccinated wild-type mice. Our results suggest that IL-10 suppresses CD4⁺ T-cell immunity in nonvaccinated mice during Coccidioides infection but does not impede the development of a memory response nor exacerbate immunopathology of vaccinated mice over at least a 4-month period after the last immunization.     

Macrophage Polarization during Murine Lyme Borreliosis

Infection of C3H mice with Borrelia burgdorferi, the causative agent of Lyme disease, reliably produces an infectious arthritis and carditis that peak around 3 weeks postinfection and then spontaneously resolve. Macrophage polarization has been suggested to drive inflammation, the clearance of bacteria, and tissue repair and resolution in a variety of infectious disease models. During Lyme disease it is clear that macrophages are capable of clearing Borrelia spirochetes and exhausted neutrophils; however, the role of macrophage phenotype in disease development or resolution has not been studied. Using classical (NOS2) and alternative (CD206) macrophage subset-specific markers, we determined the phenotype of F4/80⁺ macrophages within the joints and heart throughout the infection time course. Within the joint, CD206⁺ macrophages dominated throughout the course of infection, and NOS2⁺ macrophage numbers became elevated only during the peak of inflammation. We also found dual NOS2⁺ CD206⁺ macrophages which increased during resolution. In contrast to findings for the ankle joints, numbers of NOS2⁺ and CD206⁺ macrophages in the heart were similar at the peak of inflammation. 5-Lipoxygenase-deficient (5-LOX^−/−) mice, which display a failure of Lyme arthritis resolution, recruited fewer F4/80⁺ cells to the infected joints and heart, but macrophage subset populations were unchanged. These results highlight differences in the inflammatory infiltrates during Lyme arthritis and carditis and demonstrate the coexistence of multiple macrophage subsets within a single inflammatory site.     

Chlamydial Infection in Inducible Nitric Oxide Synthase Knockout Mice

Type 1 CD4⁺-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4⁺-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-γ)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS^+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS^−/− mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS^+/+ and iNOS^−/− mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS^−/− than in iNOS^+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS^−/− mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS^−/− mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-γ during CD4⁺-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-γ. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.