Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation.

Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11, however, are not cytosolic, and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here, we identify outer membrane vesicles (OMVs) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMVs are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMVs, reveals the importance of OMVs in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMVs in enabling the cytosolic entry of LPS and, consequently, caspase-11 activation during Gram-negative bacterial infections.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming.

Extensive desmoplasia and poor vasculature renders pancreatic tumors severely hypoxic, contributing to their aggressiveness and therapy resistance. Here, we identify the HuR/MYB/HIF1α axis as a critical regulator of the metabolic plasticity and hypoxic survival of pancreatic cancer cells. HuR undergoes nuclear-to-cytoplasmic translocation under hypoxia and stabilizes MYB transcripts, while MYB transcriptionally upregulates HIF1α. Upon MYB silencing, pancreatic cancer cells fail to survive and adapt metabolically under hypoxia, despite forced overexpression of HIF1α. MYB induces the transcription of several HIF1α-regulated glycolytic genes by directly binding to their promoters, thus enhancing the recruitment of HIF1α to hypoxia-responsive elements through its interaction with p300-dependent histone acetylation. MYB-depleted pancreatic cancer cells exhibit a dramatic reduction in tumorigenic ability, glucose-uptake and metabolism in orthotopic mouse model, even after HIF1α restoration. Together, our findings reveal an essential role of MYB in metabolic reprogramming that supports pancreatic cancer cell survival under hypoxia.

AM-301, a barrier-forming nasal spray, versus saline spray in seasonal allergic rhinitis: A randomized clinical trial.

RATIONALE: Saline nasal sprays are frequently used in the management of seasonal allergic rhinitis (SAR) for the cleansing and clearing of aeroallergens from the nasal cavity. Also using a drug-free approach, AM-301 nasal spray is forming a thin film barrier on the nasal mucosa to prevent contact with allergens, trap them, and facilitate their discharge. A clinical trial compared the efficacy, safety, and tolerability of AM-301 and saline spray in SAR. METHODS: A total of 100 patients were randomized 1:1 to self-administer AM-301 or saline 3 × daily for 2 weeks. Primary efficacy endpoint: reduction in mean daily reflective Total Nasal Symptom Score (rTNSS). Secondary efficacy endpoints: reduction in mean instantaneous TNSS and Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ), global impression of efficacy. Safety and tolerability: adverse events, relief medication use, symptom-free days, global impression of tolerability. RESULTS: AM-301-treated patients achieved a significantly lower rTNSS than the saline group (LS square means difference -1.1, 95% CI -1.959 to -0.241, p = .013) with improvement observed across all individual nasal symptoms. Likewise, all secondary endpoints showed statistical significance in favor of AM-301; for example, quality of life was significantly improved overall (p < .001) as well as for each individual RQLQ domain. Both treatments showed similarly good safety and tolerability. With AM-301, fewer patients used relief medication and more enjoyed symptom-free days compared to saline treatment. CONCLUSIONS: AM-301 was more effective than saline in improving SAR nasal symptoms and related quality of life while offering similar tolerability, demonstrating the benefits of a barrier approach.

Induction of innate immune memory via microRNA targeting of chromatin remodelling factors.

Prolonged exposure to microbial products such as lipopolysaccharide can induce a form of innate immune memory that blunts subsequent responses to unrelated pathogens, known as lipopolysaccharide tolerance. Sepsis is a dysregulated systemic immune response to disseminated infection that has a high mortality rate. In some patients, sepsis results in a period of immunosuppression (known as 'immunoparalysis')1 characterized by reduced inflammatory cytokine output2, increased secondary infection3 and an increased risk of organ failure and mortality4. Lipopolysaccharide tolerance recapitulates several key features of sepsis-associated immunosuppression5. Although various epigenetic changes have previously been observed in tolerized macrophages6-8, the molecular basis of tolerance, immunoparalysis and other forms of innate immune memory has remained unclear. Here we perform a screen for tolerance-associated microRNAs and identify miR-221 and miR-222 as regulators of the functional reprogramming of macrophages during lipopolysaccharide tolerization. Prolonged stimulation with lipopolysaccharide in mice leads to increased expression of miR-221 and mir-222, both of which regulate brahma-related gene 1 (Brg1, also known as Smarca4). This increased expression causes the transcriptional silencing of a subset of inflammatory genes that depend on chromatin remodelling mediated by SWI/SNF (switch/sucrose non-fermentable) and STAT (signal transducer and activator of transcription), which in turn promotes tolerance. In patients with sepsis, increased expression of miR-221 and miR-222 correlates with immunoparalysis and increased organ damage. Our results show that specific microRNAs can regulate macrophage tolerization and may serve as biomarkers of immunoparalysis and poor prognosis in patients with sepsis.

Immunogenicity and safety of heterologous Omicron BA.1 and bivalent SARS-CoV-2 recombinant spike protein booster vaccines: a phase 3, randomized, clinical trial.

BACKGROUND: Mutations present in emerging SARS-CoV-2 variants permit evasion of neutralization with prototype vaccines. A novel Omicron BA.1 subvariant-specific vaccine (NVX-CoV2515) was tested alone, or as a bivalent preparation in combination with the prototype vaccine (NVX-CoV2373), to assess antibody responses to SARS-CoV-2. METHODS: Participants aged 18 to 64 years immunized with 3 doses of prototype mRNA vaccines were randomized 1:1:1 to receive a single dose of NVX-CoV2515, NVX-CoV2373, or bivalent mixture in a phase 3 study investigating heterologous boosting with SARS-CoV-2 recombinant spike protein vaccines. Immunogenicity was measured 14 and 28 days after vaccination for the SARS-CoV-2 Omicron BA.1 sublineage and ancestral strain. Safety profiles of vaccines were assessed. RESULTS: Of participants who received trial vaccine (N = 829), those administered NVX-CoV2515 (n = 286) demonstrated superior neutralizing antibody response to BA.1 versus NVX-CoV2373 (n = 274) at Day 14 (geometric mean titer ratio [95% CI]: 1.6 [1.33, 2.03]). Seroresponse rates [n/N; 95% CI] were 73.4% [91/124; 64.7, 80.9] for NVX-CoV2515 versus 50.9% [59/116; 41.4, 60.3] for NVX-CoV2373. All formulations were similarly well-tolerated. CONCLUSIONS: NVX-CoV2515 elicited a superior neutralizing antibody response against the Omicron BA.1 subvariant compared with NVX-CoV2373 when administered as a fourth dose. Safety data were consistent with the established safety profile of NVX-CoV2373.

The TLR-chaperone CNPY3 is a critical regulator of NLRP3-inflammasome activation.

TLRs mediate the recognition of microbial and endogenous insults to orchestrate the inflammatory response. TLRs localize to the plasma membrane or endomembranes, depending on the member, and rely critically on ER-resident chaperones to mature and reach their subcellular destinations. The chaperone canopy FGF signaling regulator 3 (CNPY3) is necessary for the proper trafficking of multiple TLRs including TLR1/2/4/5/9 but not TLR3. However, the exact role of CNPY3 in inflammatory signalling downstream of TLRs has not been studied in detail. Consistent with the reported client specificity, we report here that functional loss of CNPY3 in engineered macrophages impairs downstream signalling by TLR2 but not TLR3. Unexpectedly, CNPY3-deficient macrophages show reduced IL-1ß and IL-18 processing and production independent of the challenged upstream TLR species, demonstrating a separate, specific role for CNPY3 in inflammasome activation. Mechanistically, we document that CNPY3 regulates caspase-1 localization to the apoptosis speck and autoactivation of caspase-1. Importantly, we were able to recapitulate these findings in macrophages from an early infantile epileptic encephalopathy (EIEE) patient with a novel CNPY3 loss-of-function variant. Summarizing, our findings reveal a hitherto unknown, TLR-independent role of CNPY3 in inflammasome activation, highlighting a more complex and dedicated role of CNPY3 to the inflammatory response than anticipated.

Distal CA1 Maintains a More Coherent Spatial Representation than Proximal CA1 When Local and Global Cues Conflict.

Entorhinal cortical projections show segregation along the transverse axis of CA1, with the medial entorhinal cortex (MEC) sending denser projections to proximal CA1 (pCA1) and the lateral entorhinal cortex (LEC) sending denser projections to distal CA1 (dCA1). Previous studies have reported functional segregation along the transverse axis of CA1 correlated with the functional differences in MEC and LEC. pCA1 shows higher spatial selectivity than dCA1 in these studies. We employ a double rotation protocol, which creates an explicit conflict between the local and the global cues, to understand the differential contributions of these reference frames to the spatial code in pCA1 and dCA1 in male Long-Evans rats. We show that pCA1 and dCA1 respond differently to this local-global cue conflict. pCA1 representation splits as predicted from the strong conflicting inputs it receives from MEC and dCA3. In contrast, dCA1 rotates more in concert with the global cues. In addition, pCA1 and dCA1 display comparable levels of spatial selectivity in this study. This finding differs from the previous studies, perhaps because of richer sensory information available in our behavior arena. Together, these observations indicate that the functional segregation along proximodistal axis of CA1 is not of the amount of spatial selectivity but that of the nature of the different inputs used to create and anchor spatial representations.SIGNIFICANCE STATEMENT Subregions of the hippocampus are thought to play different roles in spatial navigation and episodic memory. It was previously thought that the distal part of area CA1 of the hippocampus carries lesser information about space than proximal CA1 (pCA1). We report that distal CA1 (dCA1) spatial representation moves more in concert with the global cues than pCA1 when the local and the global cues conflict. We also show that spatial selectivity is comparable along the proximodistal axis in this experimental protocol. Thus, different parts of the brain receiving differential outputs from pCA1 and dCA1 receive spatial information in different spatial reference frames encoded using different sets of inputs, rather than different amounts of spatial information as thought earlier.

Nicotine causes alternative polarization of macrophages via Src-mediated STAT3 activation: Potential pathobiological implications.

Nicotine is an addictive ingredient of tobacco products and other noncigarette substitutes, including those being used for smoking cessation to relieve withdrawal symptoms. Earlier research, however, has associated nicotine with the risk and poorer outcome of several diseases, including cancer. Macrophages are an important component of the innate immune system and can have both pro-and anti-inflammatory functions depending upon their polarization state. Here, we investigated the effect of nicotine on macrophage polarization, growth, and invasion to understand its role in human physiology. We observed that nicotine induced M2 polarization of RAW264.7 and THP-1-derived macrophages in a dose-dependent manner. Cytokine profiling suggested a mixed M2a/d phenotype of nicotine-polarized macrophages associated with tissue repair and pro-angiogenic functions. Moreover, nicotine treatment also enhanced the growth, motility, and invasion of macrophages. Mechanistic studies revealed increased phosphorylation of STAT3 in nicotine-treated macrophages that was mediated through Src activation. Importantly, pretreatment of macrophages with either Src or STAT3 inhibitor abrogated nicotine-induced macrophage polarization, growth, and motility, suggesting a functional role of the Src-STAT3 signaling axis. Together, our findings reveal a novel role of nicotine in immunosuppression via causing M2 polarization of macrophages that could be implicated in the pathogenesis of various diseases.

MYB interacts with androgen receptor, sustains its ligand-independent activation and promotes castration resistance in prostate cancer.

BACKGROUND: Aberrant activation of androgen receptor signalling following castration therapy is a common clinical observation in prostate cancer (PCa). Earlier, we demonstrated the role of MYB overexpression in androgen-depletion resistance and PCa aggressiveness. Here, we investigated MYB-androgen receptor (AR) crosstalk and its functional significance. METHODS: Interaction and co-localization of MYB and AR were examined by co-immunoprecipitation and immunofluorescence analyses, respectively. Protein levels were measured by immunoblot analysis and enzyme-linked immunosorbent assay. The role of MYB in ligand-independent AR transcriptional activity and combinatorial gene regulation was studied by promoter-reporter and chromatin immunoprecipitation assays. The functional significance of MYB in castration resistance was determined using an orthotopic mouse model. RESULTS: MYB and AR interact and co-localize in the PCa cells. MYB-overexpressing PCa cells retain AR in the nucleus even when cultured under androgen-deprived conditions. AR transcriptional activity is also sustained in MYB-overexpressing cells in the absence of androgens. MYB binds and promotes AR occupancy to the KLK3 promoter. MYB-overexpressing PCa cells exhibit greater tumorigenicity when implanted orthotopically and quickly regain growth following castration leading to shorter mice survival, compared to those carrying low-MYB-expressing prostate tumours. CONCLUSIONS: Our findings reveal a novel MYB-AR crosstalk in PCa and establish its role in castration resistance.

Peritoneal Level of CD206 Associates With Mortality and an Inflammatory Macrophage Phenotype in Patients With Decompensated Cirrhosis and Spontaneous Bacterial Peritonitis.

BACKGROUND & AIMS: Peritoneal macrophages (PMs) regulate inflammation and control bacterial infections in patients with decompensated cirrhosis. We aimed to characterize PMs and associate their activation with outcomes of patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from ascites samples of 66 patients with decompensated cirrhosis (19 with SBP) and analyzed them by flow cytometry, quantitative real-time polymerase chain reaction, functional analysis, and RNA microarrays. We used ascites samples of a separate cohort of 111 patients with decompensated cirrhosis (67 with SBP) and quantified the soluble form of the mannose receptor (CD206) and tumor necrosis factor by enzyme-linked immunosorbent assay (test cohort). We performed logistic regression analysis to identify factors associated with 90-day mortality. We validated our findings using data from 71 patients with cirrhosis and SBP. Data from 14 patients undergoing peritoneal dialysis for end-stage renal disease but without cirrhosis were included as controls. RESULTS: We used surface levels of CD206 to identify subsets of large PMs (LPM) and small PMs (SPM), which differed in granularity and maturation markers, in ascites samples from patients with cirrhosis. LPMs vs SPMs from patients with cirrhosis had different transcriptomes; we identified more than 4000 genes that were differentially regulated in LPMs vs SPMs, including those that regulate the cycle, metabolism, self-renewal, and immune cell signaling. LPMs had an inflammatory phenotype, were less susceptible to tolerance induction, and released more tumor necrosis factor than SPMs. LPMs from patients with cirrhosis produced more inflammatory cytokines than LPMs from controls. Activation of PMs by Toll-like receptor agonists and live bacteria altered levels of CD206 on the surface of LPMs and release of soluble CD206. Analysis of serial ascites fluid from patients with SBP revealed loss of LPMs in the early phase of SBP, but levels increased after treatment. In the test and validation cohorts, patients with SBP and higher concentrations of soluble CD206 in ascites fluid (>0.53 mg/L) were less likely to survive for 90 days than those with lower levels. CONCLUSIONS: Surface level of CD206 can be used to identify mature, resident, inflammatory PMs in patients with cirrhosis. Soluble CD206 is released from activated LPMs and increased concentrations in patients with cirrhosis and SBP indicate reduced odds of surviving for 90 days.

Differential propagation of ripples along the proximodistal and septotemporal axes of dorsal CA1 of rats.

The functional connectivity of the hippocampus with its primary cortical input, the entorhinal cortex, is organized topographically. In area CA1 of the hippocampus, this leads to different functional gradients along the proximodistal and septotemporal axes of spatial/sensory responsivity and spatial resolution respectively. CA1 ripples, a network phenomenon, allow us to test whether the hippocampal neural network shows corresponding gradients in functional connectivity along the two axes. We studied the occurrence and propagation of ripples across the entire proximodistal axis along with a comparable spatial range of the septotemporal axis of dorsal CA1. We observed that ripples could occur at any location, and their amplitudes were independent of the tetrode location along the proximodistal and septotemporal axes. When a ripple was detected on a particular tetrode ("reference tetrode"), however, the probability of cooccurrence of ripples and ripple amplitude observed on the other tetrodes decreased as a function of distance from the reference tetrode. This reduction was greater along the proximodistal axis than the septotemporal axis. Furthermore, we found that ripples propagate primarily along the proximodistal axis. Thus, over a spatial scale of ∼1.5 mm, the network is anisotropic along the two axes, complementing the topographically organized cortico-hippocampal connections.

Inexpensive, scalable camera system for tracking rats in large spaces.

Most studies of neural correlates of spatial navigation are restricted to small arenas (≤1 m2) because of the limits imposed by the recording cables. New wireless recording systems have a larger recording range. However, these neuronal recording systems lack the ability to track animals in large area, constraining the size of the arena. We developed and benchmarked an open-source, scalable multicamera tracking system based on low-cost hardware. This "Picamera system" was used in combination with a wireless recording system for characterizing neural correlates of space in environments of sizes up to 16.5 m2. The Picamera system showed substantially better temporal accuracy than a popular commercial system. An explicit comparison of one camera from the Picamera system with a camera from the commercial system showed improved accuracy in estimating spatial firing characteristics and head direction tuning of neurons. This improved temporal accuracy is crucial for accurately aligning videos from multiple cameras in large spaces and characterizing spatially modulated cells in a large environment. NEW & NOTEWORTHY Studies of neural correlates of space are limited to biologically unrealistically small spaces by neural recording and position tracking hardware. We developed a camera system capable of tracking animals in large spaces at a high temporal accuracy. Together with the new wireless recording systems, this system facilitates the study of neural correlates of space at biologically relevant scale. This increased temporal accuracy of tracking also improves the estimates of spatiotemporal correlates of neural activity.

Streptococci Engage TLR13 on Myeloid Cells in a Site-Specific Fashion.

Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to ß-hemolytic streptococci.

Emerging evidence for the role of differential tumor microenvironment in breast cancer racial disparity: a closer look at the surroundings.

Although increased awareness leading to early detection and prevention, as well as advancements in treatment strategies, have resulted in superior clinical outcomes, African American women with breast cancer continue to have greater mortality rates, compared to Caucasian American counterparts. Moreover, African American women are more likely to have breast cancer at a younger age and be diagnosed with aggressive tumor sub-types. Such racial disparities can be attributed to socioeconomic differences, but it is increasingly being recognized that these disparities may indeed be due to certain genetic and other non-genetic biological differences. Tumor microenvironment, which provides a favorable niche for the growth of tumor cells, is comprised of several types of stromal cells and the various proteins secreted as a consequence of bi-directional tumor-stromal cross-talk. Emerging evidence suggests inherent biological differences in the tumor microenvironment of breast cancer patients from different racial backgrounds. Tumor microenvironment components, affected by the genetic make-up of the tumor cells as well as other non-tumor-associated factors, may also render patients more susceptible to the development of aggressive tumors and faster progression of disease resulting in early onset, thus adversely affecting patients' survival. This review provides an overview of breast cancer racial disparity and discusses the existence of race-associated differential tumor microenvironment and its underlying genetic and non-genetic causal factors. A better understanding of these aspects would help further research on effective cancer management and improved approaches for reducing the racial disparities gaps in breast cancer patients.

Clostridium perfringens beta toxin DNA prime-protein boost elicits enhanced protective immune response in mice.

Clostridium perfringens beta toxin (CPB) is the primary pathogenic factor responsible for necrotic enteritis in sheep, cattle and humans. Owing to rapid progression of the disease, vaccination is the only possible recourse to avoid high mortality in animal farms and huge economic losses. The present study reports evaluation of a cpb gene-based DNA vaccine encoding the beta toxin of C. perfringens with homologous as well as heterologous booster strategy. Immunization strategy employing heterologous booster with heat-inactivated rCPB mounted stronger immune response when compared to that generated by homologous booster. Antibody isotyping and cytokine ELISA demonstrated the immune response to be Th1-biased mixed immune response. While moderate protection of immunized BALB/c and C57BL/6 mice against rCPB challenge was observed with homologous booster strategy, heterologous booster strategy led to complete protection. Thus, beta toxin-based DNA vaccine using the heterologous prime-boosting strategy was able to generate better immune response and conferred greater degree of protection against high of dose rCPB challenge than homologous booster regimen, making it an effective vaccination approach against C. perfringens beta toxin.

The endolysosomal cysteine cathepsins L and K are involved in macrophage-mediated clearance of Staphylococcus aureus and the concomitant cytokine induction.

Cysteine cathepsins are endolysosomal cysteine proteases highly expressed in macrophages; however, their individual contributions to the elimination of bacteria and bacteria-induced cytokine production by macrophages are unknown. We assessed the contribution of cysteine cathepsins to macrophage defense pathways against Staphylococcus aureus by using chemical inhibitors and by infecting primary bone marrow-derived macrophages deficient in 1 of 7 major macrophage-expressed endolysosomal cysteine proteases. We show that cysteine cathepsins are involved in the phagocytosis and killing of S. aureus. Cathepsin L was identified as an executor of nonoxidative killing. Moreover, microarray data revealed cysteine cathepsins to be important for the maximal induction of certain proinflammatory genes, such as IL6, in response to S. aureus. Cysteine cathepsin's contribution to IL6 production was dependent on phagocytosis, and cathepsin K was identified to be a critical protease in this process. Analysis of macrophages with impaired trafficking of endolysosomal Toll-like receptors (TLRs) to the acidic compartment revealed that they were not involved in cathepsin-dependent IL6 induction. Because IL6 production was completely dependent on the TLR-adaptor protein myeloid differentiation primary response gene 88 (MyD88), it appears that other TLRs are involved. In summary, lysosomal cysteine proteases are functionally linked to the complex bactericidal and inflammatory activities of macrophages.