Disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with high mobility and mortality. However, the molecular basis of host defense against disseminated candidiasis remains elusive, and treatment options are limited. Here we report that the E3 ubiquitin ligase CBLB directs polyubiquitination of dectin-1 and dectin-2, two key pattern-recognition receptors for sensing Candida albicans, and their downstream kinase SYK, thus inhibiting dectin-1- and dectin-2-mediated innate immune responses. CBLB deficiency or inactivation protects mice from systemic infection with a lethal dose of C. albicans, and deficiency of dectin-1, dectin-2, or both in Cblb(-/-) mice abrogates this protection. Notably, silencing the Cblb gene in vivo protects mice from lethal systemic C. albicans infection. Our data reveal that CBLB is crucial for homeostatic control of innate immune responses mediated by dectin-1 and dectin-2. Our data also indicate that CBLB represents a potential therapeutic target for protection from disseminated candidiasis.

Sepsis patients with cardiac dysfunction have significantly higher mortality. Although several pathways are associated with myocardial damage in sepsis, the precise cause(s) remains unclear and treatment options are limited. This study was designed to develop a new model to investigate the early events of cardiac damage during sepsis progression.

Peroxisome proliferator-activated receptor (PPAR)γ is a global transcriptional regulator associated with anti-inflammatory actions. It is highly expressed in alveolar macrophages (AMs), which are unable to clear the intracellular pathogen Mycobacterium tuberculosis (M.tb). Although M.tb infection induces PPARγ in human macrophages, which contributes to M.tb growth, the mechanisms underlying this are largely unknown. We undertook NanoString gene expression analysis to identify novel PPARγ effectors that condition macrophages to be more susceptible to M.tb infection. This revealed several genes that are differentially regulated in response to PPARγ silencing during M.tb infection, including the Bcl-2 family members Bax (pro-apoptotic) and Mcl-1 (pro-survival). Apoptosis is an important defense mechanism that prevents the growth of intracellular microbes, including M.tb, but is limited by virulent M.tb. This suggested that M.tb differentially regulates Mcl-1 and Bax expression through PPARγ to limit apoptosis. In support of this, gene and protein expression analysis revealed that Mcl-1 expression is driven by PPARγ during M.tb infection in human macrophages. Further, 15-lipoxygenase (15-LOX) is critical for PPARγ activity and Mcl-1 expression. We also determined that PPARγ and 15-LOX regulate macrophage apoptosis during M.tb infection, and that pre-clinical therapeutics that inhibit Mcl-1 activity significantly limit M.tb intracellular growth in both human macrophages and an in vitro TB granuloma model. In conclusion, identification of the novel PPARγ effector Mcl-1 has determined PPARγ and 15-LOX are critical regulators of apoptosis during M.tb infection and new potential targets for host-directed therapy for M.tb.

Complement receptor 3 (CR3, CD11b/CD18) is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs) requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting MAPK activation through outside-in signaling. CR3-linked immune suppression is an important mechanism involved in the pathogenesis of F. tularensis infection.

The intracellular gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3'UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFkappaB. Consistently, macrophages lacking SHIP displayed enhanced NFkappaB-driven gene transcription, whereas overexpression of SHIP led to decreased NFkappaB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFkappaB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.

Activation, recruitment, and effector function of T lymphocytes are essential for control of mycobacterial infection. These processes are tightly regulated in T cells by the availability of l-arginine within the microenvironment. In turn, mycobacterial infection dampens T cell responsiveness through arginase induction in myeloid cells, promoting sequestration of l-arginine within the local milieu. Here, we show T cells can replenish intracellular l-arginine through metabolism of l-citrulline to mediate inflammatory function, allowing anti-mycobacterial T cells to overcome arginase-mediated suppression. Furthermore, T cell l-citrulline metabolism is necessary for accumulation of CD4+ T cells at the site of infection, suggesting this metabolic pathway is involved during anti-mycobacterial T cell immunity in vivo. Together, these findings establish a contribution for l-arginine synthesis by T cells during mycobacterial infection, and implicate l-citrulline as a potential immuno-nutrient to modulate host immunity.

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.

SARS-CoV-2 virus has infected more than 92 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Using a rhesus macaque model of SARS-CoV-2 infection, we have characterized the transcriptional signatures induced in the lungs of juvenile and old macaques following infection. Genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated, as also seen in lungs of macaques with tuberculosis. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. Together, our transcriptomic studies have delineated disease pathways that improve our understanding of the immunopathogenesis of COVID-19.

Aging increases the risk of tuberculosis (TB) and its adverse outcomes, but most studies are based on secondary analyses, and few are in Hispanics. Diabetes is a risk factor for TB in adults, but its contribution in the elderly is unknown. We aimed to identify the role of diabetes and other risk factors for TB in elderly Hispanics.

Mycobacterium indicus pranii (MIP) known for its immunotherapeutic potential against leprosy and tuberculosis is undergoing various clinical trials and also simultaneously being studied in animal models to get insight into the mechanistic details contributing to its protective efficacy as a vaccine candidate. Studies have shown potential immunomodulatory properties of MIP, the most significant being the ability to induce strong Th1 type of response, enhanced expression of pro-inflammatory cytokines, activation of APCs and lymphocytes, elicitation of M.tb specific poly-functional T cells. All of these form crucial components of host-immune response during M.tb infection. Also, MIP was found to be potent inducer of autophagy in macrophages which resulted in enhanced clearance of M.tb from MIP and M.tb co-infected cells. Hence, we further examined the component/s of MIP responsible for autophagy induction. Interestingly, we found that MIP lipids and DNA were able to induce autophagy but not the protein fraction. LAM being one of the crucial components of mycobacterial cell-wall lipids and possessing the ability of immunomodulation; we isolated LAM from MIP and did a comparative study with M.tb-LAM. Stimulation with MIP-LAM resulted in significantly high secretion of pro-inflammatory cytokines and displayed high autophagy inducing potential in macrophages as compared to M.tb-LAM. Treatment with MIP-LAM enhanced the co-localization of M.tb within the phago-lysosomes and increased the clearance of M.tb from the infected macrophages. This study describes LAM to be a crucial component of MIP which has significant contribution to its immunotherapeutic efficacy against TB.

Age is a major risk factor for chronic infections, including tuberculosis (TB). Elderly TB patients also suffer from elevated levels of psychological stress. It is not clear how psychological stress impacts immune response to Mycobacterium tuberculosis (M.tb). In this study, we used social disruption stress (SDR) to investigate effects of psychological stress in young and old mice. Unexpectedly, we found that SDR suppresses lung inflammation in old mice as evidenced by lower pro-inflammatory cytokine levels in bronchial lavage fluid and decreased cytokine mRNA expression by alveolar macrophages. To investigate effects of stress on M.tb infection, mice were subjected to SDR and then infected with M.tb. As previously reported, old mice were better at controlling infection at 30 days than young mice. This control was transient as CFUs at 60 days were higher in old control mice compared to young mice. Consistently, SDR significantly increased M.tb growth at 60 days in old mice compared to young mice. In addition, SDR in old mice resulted in accumulation of IL-10 mRNA and decreased IFN-γ mRNA at 60 days. Also, confocal microscopy of lung sections from old SDR mice showed increased number of CD4 T cells which express LAG3 and CD49b, markers of IL-10 secreting regulatory T cells. Further, we also demonstrated that CD4 T cells from old SDR mice express IL-10. Thus, we conclude that psychological stress in old mice prior to infection, increases differentiation of IL-10 secreting T cells, which over time results in loss of control of the infection.

Tuberculosis (TB) accounts for 1.6 million deaths annually and over 25% of deaths due to antimicrobial resistance. Mycobacterium tuberculosis (M.tb) drives MCL-1 expression (family member of anti-apoptotic BCL-2 proteins) to limit apoptosis and grow intracellularly in human macrophages. The feasibility of re-purposing specific MCL-1 and BCL-2 inhibitors to limit M.tb growth, using inhibitors that are in clinical trials and FDA-approved for cancer treatment has not be tested previously. We show that specifically inhibiting MCL-1 and BCL-2 induces apoptosis of M.tb-infected macrophages, and markedly reduces M.tb growth in human and murine macrophages, and in a pre-clinical model of human granulomas. MCL-1 and BCL-2 inhibitors limit growth of drug resistant and susceptible M.tb in macrophages and act in additive fashion with the antibiotics isoniazid and rifampicin. This exciting work uncovers targeting the intrinsic apoptosis pathway as a promising approach for TB host-directed therapy. Since safety and activity studies are underway in cancer clinics for MCL-1 and BCL-2 inhibitors, we expect that re-purposing them for TB treatment should translate more readily and rapidly to the clinic. Thus, the work supports further development of this host-directed therapy approach to augment current TB treatment.

MiR-155 regulates numerous aspects of innate and adaptive immune function. This miR is induced in response to Toll-like receptor ligands, cytokines, and microbial infection. We have previously shown that miR-155 is induced in monocytes/macrophages infected with Francisella tularensis and suppresses expression of the inositol phosphatase SHIP to enhance activation of the PI3K/Akt pathway, which in turn promotes favorable responses for the host. Here we examined how miR-155 expression is regulated during infection. First, our data demonstrate that miR-155 can be induced through soluble factors of bacterial origin and not the host. Second, miR-155 induction is not a direct effect of infection and it requires NF-κB signaling to up-regulate fos/jun transcription factors. Finally, we demonstrate that the requirement for NF-κB-dependent de novo protein synthesis is globally shared by microbial ligands and live bacteria. This study provides new insight into the complex regulation of miR-155 during microbial infection.

The exact role of CD8(+) T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8(+) T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8(+) T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1(+), Tim-3(+), CD122(+)). CD8(+) T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8(+) T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8(+) T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8(+) CD8(+) T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8(+) did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8(+) T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.

Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B-cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB-exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti-MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies.

Biological aging dynamically alters normal immune and cardiac function, favoring the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased instances of cardiac distress. Cardiac failure is the primary reason for hospitalization of the elderly (65+ years). The elderly are also increasingly susceptible to developing chronic bacterial infections due to aging associated immune abnormalities. Since bacterial infections compound the rates of cardiac failure in the elderly, and this phenomenon is not entirely understood, the interplay between the immune system and cardiovascular function in the elderly is of great interest. Using Mycobacterium avium, an opportunistic pathogen, we investigated the effect of mycobacteria on cardiac function in aged mice. Young (2-3 months) and old (18-20 months) C57BL/6 mice were intranasally infected with M. avium strain 104, and we compared the bacterial burden, immune status, cardiac electrical activity, pathology, and function of infected mice against uninfected age-matched controls. Herein, we show that biological aging may predispose old mice infected with M. avium to mycobacterial dissemination into the heart tissue and this leads to cardiac dysfunction. M. avium infected old mice had significant dysrhythmia, cardiac hypertrophy, increased recruitment of CD45+ leukocytes, cardiac fibrosis, and increased expression of inflammatory genes in isolated heart tissue. This is the first study to report the effect of mycobacteria on cardiac function in an aged model. Our findings are critical to understanding how nontuberculous mycobacterium (NTM) and other mycobacterial infections contribute to cardiac dysfunction in the elderly population.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPβ, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

While the advent of combination antiretroviral therapy (ART) has significantly improved survival, tuberculosis (TB) remains the leading cause of death in the HIV-infected population. We used Mycobacterium tuberculosis/simian immunodeficiency virus-coinfected (M. tuberculosis/SIV-coinfected) macaques to model M. tuberculosis/HIV coinfection and study the impact of ART on TB reactivation due to HIV infection. Although ART significantly reduced viral loads and increased CD4+ T cell counts in blood and bronchoalveolar lavage (BAL) samples, it did not reduce the relative risk of SIV-induced TB reactivation in ART-treated macaques in the early phase of treatment. CD4+ T cells were poorly restored specifically in the lung interstitium, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction in myeloid cells in the inducible bronchus-associated lymphoid tissue (iBALT) likely contributed to dysregulated T cell homing and impaired lung immunity. Thus, although ART was indispensable for controlling viral replication, restoring CD4+ T cells, and preventing opportunistic infection, it appeared inadequate in reversing the clinical signs of TB reactivation during the relatively short duration of ART administered in this study. This finding warrants the modeling of concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV to inform treatment strategies in patients with M. tuberculosis/HIV coinfection.