Interactions between B cells and T follicular regulatory cells enhance susceptibility to Brucella infection independent of the anti-Brucella humoral response.

Brucellosis, caused by facultative, intracellular Brucella spp., often results in chronic and/or lifelong infection. Therefore, Brucella must employ mechanisms to subvert adaptive immunity to cause chronic infection. B lymphocytes enhance susceptibility to infection with Brucella spp. though the mechanisms remain unclear. Here we investigated the role of antibody secretion, B cell receptor (BCR) specificity, and B cell antigen presentation on susceptibility to B. melitensis. We report that mice unable to secrete antibody do not display altered resistance to Brucella. However, animals with B cells that are unable to recognize Brucella through their BCR are resistant to infection. In addition, B cell MHCII expression enhances susceptibility to infection in a CD4+ T cell-dependent manner, and we found that follicular B cells are sufficient to inhibit CD4+ T cell-mediated immunity against Brucella. B cells promote development of T follicular helper (TFH) and T follicular regulatory (TFR) cells during Brucella infection. Inhibition of B cell and CD4+ T cell interaction via CD40L blockade enhances resistance to Brucella in a B cell dependent manner concomitant with suppression of TFH and TFR differentiation. Conversely, PD-1 blockade increases Brucella burdens in a B and CD4+ T cell dependent manner while augmenting T regulatory (TReg) and TFR responses. Intriguingly, TFR deficiency enhances resistance to Brucella via a B cell dependent, but antibody independent mechanism. Collectively, these results demonstrate B cells support TFR responses that promote susceptibility to Brucella infection independent of the antibody response.

Innate Lymphoid Cells and Interferons Limit Neurologic and Articular Complications of Brucellosis.

Brucellosis is a globally significant zoonotic disease. Human patients with brucellosis develop recurrent fever and focal complications, including arthritis and neurobrucellosis. The current study investigated the role of innate lymphoid cells (ILCs) in the pathogenesis of focal brucellosis caused by Brucella melitensis. After footpad infection, natural killer cells and ILC1 cells both limited joint colonization by Brucella. Mice lacking natural killer cells, and in particular mice lacking all ILCs, also developed marked arthritis after footpad infection. Following pulmonary infection, mice lacking adaptive immune cells and ILCs developed arthritis, neurologic complications, and meningitis. Adaptive immune cells and ILCs both limited colonization of the brain by Brucella following pulmonary infection. Transcriptional analysis of Brucella-infected brains revealed marked up-regulation of genes associated with inflammation and interferon responses, as well as down-regulation of genes associated with neurologic function. Type II interferon deficiency resulted in colonization of the brain by Brucella, but mice lacking both type I and type II interferon signaling more rapidly developed clinical signs of neurobrucellosis, exhibited hippocampal neuronal loss, and had higher levels of Brucella in their brains than mice lacking type II interferon signaling alone. Collectively, these findings indicate ILCs and interferons play an important role in prevention of focal complications during Brucella infection, and that mice with deficiencies in ILCs or interferons can be used to study pathogenesis of neurobrucellosis.

Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

MyD88-Dependent Glucose Restriction and Itaconate Production Control Brucella Infection.

Brucellosis is one of the most common global zoonoses and is caused by facultative intracellular bacteria of the genus Brucella. Numerous studies have found that MyD88 signaling contributes to protection against Brucella; however, the underlying mechanism has not been entirely defined. Here, we show that MyD88 signaling in hematopoietic cells contributes both to inflammation and to control of Brucella melitensis infection in vivo. While the protective role of MyD88 in Brucella infection has often been attributed to promotion of gamma interferon (IFN-γ) production, we found that MyD88 signaling restricts host colonization by B. melitensis even in the absence of IFN-γ. In vitro, we show that MyD88 promotes macrophage glycolysis in response to B. melitensis. Interestingly, a B. melitensis mutant lacking the glucose transporter, GluP, was more highly attenuated in MyD88-/- than in wild-type mice, suggesting MyD88 deficiency results in an increased availability of glucose in vivo, which Brucella can exploit via GluP. Metabolite profiling of macrophages identified several metabolites regulated by MyD88 in response to B. melitensis, including itaconate. Subsequently, we found that itaconate has antibacterial effects against Brucella and also regulates the production of proinflammatory cytokines in B. melitensis-infected macrophages. Mice lacking the ability to produce itaconate were also more susceptible to B. melitensis in vivo. Collectively, our findings indicate that MyD88-dependent changes in host metabolism contribute to control of Brucella infection.

Nitric oxide inhibits interleukin-1-mediated protection against Escherichia coli K1-induced sepsis and meningitis in a neonatal murine model.

Neonatal meningitis-associated Escherichia coli (NMEC) is a leading cause of sepsis and meningitis in newborn infants. Neonates are known to have impaired inflammasome activation and interleukin (IL)-1 production. However, it is unknown what role this plays in the context of NMEC infection. Here we investigated the role of IL-1 signaling in the pathogenesis of NMEC infection. We found both IL-1ß and IL-1α were secreted from macrophages and microglial cells in response to NMEC in a Toll-like receptor 4- and NLR family pyrin domain containing 3 (NPLR3)-dependent manner. Intracerebral infection of adult mice indicated a protective role of IL-1 signaling during NMEC infection. However, IL-1 receptor blockade in wild-type neonatal mice did not significantly alter bacterial loads in the blood or brain, and we, therefore, investigated whether protection conferred by IL-1 was age dependent. Neonates are known to have increased nitric oxide (NO) levels compared with adults, and we found NO inhibited the secretion of IL-1 by macrophages in response to NMEC. In contrast to our results in wild-type neonates, blockade of IL-1 receptor in neonates lacking inducible nitric oxide synthase (iNOS) led to significantly increased bacterial loads in the blood and brain. These data indicate IL-1 signaling is protective during NMEC infection in neonates only when iNOS is absent. Collectively, our findings suggest that increased NO production by neonates inhibits IL-1 production, and that this suppresses the protective role of IL-1 signaling in response to NMEC infection. This may indicate a general mechanism for increased susceptibility of neonates to infection and could lead to new therapeutic strategies in the future.

B Cells Inhibit CD4+ T Cell-Mediated Immunity to Brucella Infection in a Major Histocompatibility Complex Class II-Dependent Manner.

Brucella spp. are facultative intracellular bacteria notorious for their ability to induce a chronic, and often lifelong, infection known as brucellosis. To date, no licensed vaccine exists for prevention of human disease, and mechanisms underlying chronic illness and immune evasion remain elusive. We and others have observed that B cell-deficient mice challenged with Brucella display reduced bacterial burden following infection, but the underlying mechanism has not been clearly defined. Here, we show that at 1 month postinfection, B cell deficiency alone enhanced resistance to splenic infection ∼100-fold; however, combined B and T cell deficiency did not impact bacterial burden, indicating that B cells only enhance susceptibility to infection when T cells are present. Therefore, we investigated whether B cells inhibit T cell-mediated protection against Brucella Using B and T cell-deficient Rag1-/- animals as recipients, we demonstrate that adoptive transfer of CD4+ T cells alone confers marked protection against Brucella melitensis that is abrogated by cotransfer of B cells. Interestingly, depletion of CD4+ T cells from B cell-deficient, but not wild-type, mice enhanced susceptibility to infection, further confirming that CD4+ T cell-mediated immunity against Brucella is inhibited by B cells. In addition, we found that the ability of B cells to suppress CD4+ T cell-mediated immunity and modulate CD4+ T cell effector responses during infection was major histocompatibility complex class II (MHCII)-dependent. Collectively, these findings indicate that B cells modulate CD4+ T cell function through an MHCII-dependent mechanism which enhances susceptibility to Brucella infection.

IFN-γ-dependent nitric oxide suppresses Brucella-induced arthritis by inhibition of inflammasome activation.

Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a globally important zoonotic disease for which arthritis is the most common focal complication in humans. Wild-type mice infected systemically with Brucella typically do not exhibit arthritis, but mice lacking IFN-γ develop arthritis regardless of the route of Brucella infection. Here, we investigated mechanisms by which IFN-γ suppresses Brucella-induced arthritis. Several cell types, including innate lymphoid cells, contributed to IFN-γ production and suppression of joint swelling. IFN-γ deficiency resulted in elevated joint IL-1ß levels, and severe joint inflammation that was entirely inflammasome dependent, and in particular, reliant on the NLRP3 inflammasome. IFN-γ was vital for induction of the nitric oxide producing enzyme, iNOS, in infected joints, and nitric oxide directly inhibited IL-1ß production and inflammasome activation in Brucella-infected macrophages in vitro. During in vivo infection, iNOS deficiency resulted in an increase in IL-1ß and inflammation in Brucella-infected joints. Collectively, this data indicate that IFN-γ prevents arthritis both by limiting Brucella infection, and by inhibiting excessive inflammasome activation through the induction of nitric oxide.