Pam3 CSK4 Research Articles

Delineating Brainstem CircRNAs role in Neonatal sepsis

Background: Neonatal sepsis remains one of the leading causes of mortality in newborns. Despite advances in newborn care, the literature still deprived of mechanistic insights into neonatal sepsis. Our prior findings revealed that systemic inflammation induced by TLR4 (LPS) or TLR1/2 (PAM3CKS4) ligands destabilizes breathing in neonates and impairs inspiratory rhythmogenesis following hypoxia. Circular RNAs (circRNAs) are emerging as important mechanistic mediators in neuronal activity. CircRNA constitutes a class of closed non-coding RNAs with a closed-loop structure. Brain-expressed circRNAs are enriched during synaptogenesis and many, occurs independently of the linear transcripts. Given the abundance of circRNAs in the brain, understanding their potential functional role in health and disease represents an emerging need. Our study delineates changes that neonatal sepsis evokes on brainstem circRNAs and their epigenetic modifications. Methods:CD1 mice were i.p. treated at postnatal day 5 (PD5) with LPS, or PAM3CSK4 (PAM), or saline (control). Following euthanasia, brainstems were dissected out and kept in -80C until further processing. Total RNAs were digested with Rnase R to remove linear RNAs. Labeled circRNAs were hybridized onto the Arraystar Mouse circRNA Array V2 or Mouse circRNA Epitranscriptomic Arrays and scanned by the Agilent Scanner G2505C. All data analyses were performed in RStudio Version 2022. Results: Our data revealed, in total, 13890 circRNAs expressed in the brainstem of PD5 pups. We found hundreds of circRNAs differentially expressed (DE) as a result of systemic inflammation. Strikingly, LPS induced a very low level of circRNA modulation, whereas PAM3CSK4 enriched 184 circRNAs relative to controls. Gene ontology (GO) analysis demonstrated the enrichment of mitochondrial-metabolic processes, translation, t-RNA, and RNA aminoacylation due to LPS-inducing inflammation. PAM3CSK4 administration led enrichment of processes associated with neurogenesis, neuronal transmission, and synapse organization. Additionally, KEGG pathways identified synapse processes, including GABAergic, dopaminergic, and glutamatergic enriched by circRNAs upregulated in PAM3CSK4. Epigenetic brainstem circRNA modifications in neonatal sepsis were found due to either LPS or PAM3CSK4. A greater number of hypomethylated than hypermethylated m6A and m5c circRNA modifications were promoted by PAM3CSK4. LPS augmented hypermethylation of m6A and hypomethylation of m5c modifications. Conclusions: Brainstem expressed circRNAs were specifically regulated by the inflammatory stimuli. Neonatal sepsis increases susceptibility to circRNAs modulation in brainstem neuronal processes. Our findings indicate that circRNAs are positioned to regulate brainstem-related functions. NIH/NHLBI R01HL132355 and GF316909 Mangurian Foundation Neuro Research for JJO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Trained innate immunity modulates osteoblast and osteoclast differentiation

Macrophages are key regulators in bone repair and regeneration. Recent studies have shown that long-term epigenetic changes and metabolic shifts occur during specific immune training of macrophages that affect their functional state, resulting in heightened (trained) or reduced (tolerant) responses upon exposure to a second stimulus. This is known as innate immune memory. Here, we study the impact of macrophages’ memory trait on osteoblast differentiation of human mesenchymal stromal cells (hMSCs) and osteoclast differentiation. An in vitro trained immunity protocol of monocyte-derived macrophages was employed using inactivated Candida albicans and Bacillus Calmette–Guérin (BCG) to induce a ‘trained’ state and Pam3CSK4 (PAM) and Lipopolysaccharides (LPS) to induce a ‘tolerance’ state. Macrophages were subsequently cocultured with hMSCs undergoing osteogenic differentiation during either resting (unstimulated) or inflammatory conditions (restimulated with LPS). Alkaline phosphatase activity, mineralization, and cytokine levels (TNF, IL-6, oncostatin M and SDF-1α) were measured. In addition, macrophages underwent osteoclast differentiation. Our findings show that trained and tolerized macrophages induced opposing results. Under resting conditions, BCG-trained macrophages enhanced ALP levels (threefold), while under inflammatory conditions this was found in the LPS-tolerized macrophages (fourfold). Coculture of hMSCs with trained macrophages showed mineralization while tolerized macrophages inhibited the process under both resting and inflammatory conditions. While osteoclast differentiation was not affected in trained-macrophages, this ability was significantly loss in tolerized ones. This study further confirms the intricate cross talk between immune cells and bone cells, highlighting the need to consider this interaction in the development of personalized approaches for bone regenerative medicine.Graphical

Detailed Mechanisms Underlying Neutrophil Bactericidal Activity against Streptococcus pneumoniae.

Neutrophils are an essential cellular component of innate immunity and control bacterial infections through a combination of intracellular and extracellular killing methods. Although the importance of neutrophils has been established, the exact methods used to handle particular bacterial challenges and the efficiency of bacterial killing remain not well understood. In this study, we addressed how neutrophils eliminate Streptococcus pneumoniae (Spn), a leading cause of community acquired and post-influenza bacterial pneumonia. We analyzed killing methods with variable bacterial:neutrophil concentrations and following priming with PAM3CSK4 (P3CSK), an agonist for Toll-like-receptor 2 (TLR2). Our results show that murine neutrophils display surprisingly weak bactericidal activity against Spn, employing a predominantly extracellular mode of killing at lower concentrations of bacteria, whereas challenges with higher bacterial numbers induce both extracellular and intracellular elimination modes but require TLR2 activation. TLR2 activation increased reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation in response to Spn. Despite this, supernatants from P3CSK-stimulated neutrophils failed to independently alter bacterial replication. Our study reveals that unstimulated neutrophils are capable of eliminating bacteria only at lower concentrations via extracellular killing methods, whereas TLR2 activation primes neutrophil-mediated killing using both intracellular and extracellular methods under higher bacterial burdens.

An AP-3-dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.

Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.

Metabolic trade‐off and adipose tissue role in Neonatal Sepsis

BackgroundNeonatal sepsis contributes substantially to neonatal morbidity and mortality. The activation of an inflammatory response by the host triggers a significant metabolic challenge in which mobilization of stored energy is redistributed. Metabolic costs of immunity are high and physiological trade‐offs that neonates suffering from sepsis undergoes can determine the disease tolerance. Adipose tissue (AT) is a highly dynamic endocrine organ that partakes inter‐organs communication and regulates metabolism. In this study, we investigated whether immunity activation due to distinct TLRs ligands in neonatal sepsis promoted an energetic trade‐off and if the AT depots promoted tissue tolerance.MethodsPostnatal day 5 (PD5) CD1 mice were I.P. treated either with saline (Control), LPS, or Pam3CSK4 (PAM). Body weight and temperature were accessed prior treatments and after 3 hours. Oxygen and CO2 consumption was assessed through Comprehensive Lab Animal Monitoring System (CLAMS). Following euthanasia, blood was collected and subcutaneous white AT (WAT) and brown AT (BAT) depots were dissected out and submitted to microarray analysis utilizing Clariom™ S Assay mouse and the Affymetrix GeneChip Mouse Gene ST 2.0 arrays. Blood glucose was assessed using a glucometer.ResultsThe activation of inflammation promoted either by LPS or PAM resulted in significantly reduced total body weight due to the treatment only after 3 hours. We asked if this effect was due to the growth or nutritional status, we found the control pups kept with the mom increased body weight, whereas the pups fasting did not, as expected. However, these changes were not observed in the TLR4 and TLR1/2 ligands treated pups, in which, the bodyweight was decreased independently of them being fasted or not. The cutaneous temperature remained similar in the control group, while LPS induced a loss of ‐1.5°C degrees post‐treatment, and only ‐0.5°C variation was observed in the PAM treated pups. A reduction in VO2 and VCO2 consumption was stronger in LPS than in PAM treated pups, in which they started to increase these levels after 90 minutes, indicating that TLR4 induced energy conservation also in neonates. LPS and PAM induced diminished blood glucose levels compared to control in fasting pups, and only in LPS in fed pups. Next, we evaluated the contribution of AT to lower metabolic rate and promote energy conservation. Microarray analysis showed LPS induced a total of 103 up‐regulated genes, and 238 due to PAM in BAT. Enrichment analysis of this depot was related to Collagen‐containing extracellular matrix, and Inflammatory response in LPS treated pups, while mRNA metabolic process was highlighted in pups treated with PAM. Moreover, in the WAT, LPS promoted enrichment of inflammatory responses along with lipid storage, fatty acid‐binding and lipid metabolism, whereas PAM induced several pathways related to lipid storage, catabolism, and temperature homeostasis.ConclusionsCollectively, these data highlight the physiological trade‐offs occurring between immunity and energy suggesting that the hypometabolic‐hypothermic state is inducing energy conservation. AT depots are promoting fatty acid oxidation and perhaps mediating this energetic trade‐off. Thus, these physiological outcomes demonstrate the importance of AT regulation over the course of neonatal sepsis.

Dissecting breathing control dysregulation in neonatal sepsis

BackgroundSepsis is a common and frequently fatal condition affecting neonates. The global burden of neonatal sepsis is underappreciated especially regarding breathing and maintenance of adequate ventilation which is essential to life. Systemic inflammation induced by sepsis triggers activation of immune cells peripherally, which can be sensed and further propagated by multiple cell types in the CNS, including those in the brainstem involved in the neural control of breathing. Understanding how neonatal sepsis alters breathing plasticity is crucial for promoting earlier diagnosis and better therapeutic interventions. Here, we investigated how systemic inflammation caused by distinct TLRs ligands in neonates affects breathing plasticity.MethodsCD1 mice were I.P. treated at postnatal day 5 (PD5) with saline (control), LPS, or Pam3CSK4 (PAM), and after 3 hours, they were either euthanized or submitted to respiratory tests using Buxco unrestrained whole‐body plethysmography (WBP). After euthanasia, brainstem was dissected out and slices containing PreBötC and hypoglossal pool were prepared and electrophysiological recordings were performed simultaneously under baseline and hypoxic (95%N2/5%CO2) conditions. Microarray analysis of whole brainstem and isolated neurons, astrocytes, and microglia were performed with Affymetrix Mouse Gene ST2.0 arrays.ResultsRespiratory response to hypoxia, hypercapnia and, anoxia gas challenges were preserved and functional in neonatal PD5 pups. The ventilatory response in TLR4 and TLR1/2 ligands treated pups was reduced during baseline, and an inhibited response to hypoxic and hypercapnic conditions was observed. Recovery time after each anoxic episode was calculated as gasping latency and PAM treated pups demonstrated a disrupted ability to recover after anoxic episodes. The central respiratory activity measured in isolated preBötC slices from PAM treated pups showed that networks failed to transmit rhythmic activity to the hypoglossal nucleus. In PAM treated networks hypoxic rhythmogenesis from the preBötC was weaker, yet in network from LPS treated pups, hypoxic rhythmogenesis continued. Next, we assessed gene modulation in the brainstem PD5 pups. The majority of differentially expressed genes (DEGs) were related to innate immunity in both LPS or PAM groups. The enrichment analysis showed activation of T helper cells, whereas CIBERSORT analysis indicated enrichment of Treg and B cells memory. The molecular pathways activated in neurons, astrocytes, and microglia were founded distinct and specific to the TLR ligand. Neurons were strongly modulated in both of the groups. Surprisingly, astrocytes coming from LPS treated pups exhibited only a few DEGs whereas PAM did not induce any modulation.ConclusionsTaken together, these results shows that the ventilatory response is detrimentally affected in neonatal sepsis within only a few hours of an inflammatory insult. Not only the ability to sense O2/CO2 levels and to respond accordingly is disrupted, but also the central respiratory rhythmic activity in preBötC slices. Both TLRs ligands induced a strong innate immune response in the brainstem which is amplified in neurons especially in pups treated with TLR1/2 ligand.

Oral Tolerance Induced by Heat Shock Protein 65-Producing Lactococcus lactis Mitigates Inflammation in Leishmania braziliensis Infection.

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.

Dysregulation of breathing control through distinct molecular mechanisms of the brainstem cell population in neonatal sepsis

Introduction Neonatal sepsis is triggered by a dysregulated immune response leading to systemic inflammation and life-threatening organ dysfunction. The systemic alterations and activation of immune cells will impact the activity of neural circuits, neural and non-neuronal cell populations, including those in the brainstem involved in breathing rhythm generation and pattern. Elucidating the extent to which inflammation modulates the CNS control of breathing has eluded us due to the lack of appropriate physiological maneuvers capable of interrogating specific brainstem circuits in the neonatal period. We perform novel respiratory testing in newborn pups with unrestrained plethysmography using distinct TLR ligands to test the hypothesis that distinct forms of systemic inflammation modulate brainstem circuits in different ways. Our goal was to investigate how different systemic inflammatory insults at the neonatal period affect the brainstem by determining the contribution of neurons, astrocytes, and microglia in the breathing plasticity response. Methods CD1 mice were I.P. treated at postnatal day 5 (PD5) with LPS, or Pam3CSK4(PAM), or saline (control). Using a whole-body plethysmography system, we assessed brainstem arousal reflex. Briefly, unrestrained pups were placed inside of the chamber individually, and submitted to a room air (RA) exposure for 06 min, followed by 01 min of a gas mixture of 97%N2/3%CO2 (anoxia) repeated 05 times. Ultrasonic vocalization recordings, a measure of brainstem-spinal circuit prolonged-forced expiration, were obtained with the Avisoft SASLab Pro software. Bulk brainstem and gene expression analysis of isolated neurons, astrocytes, and microglia were performed with Clariom™ S Assay mouse and Affymetrix GeneChip Mouse Gene ST 2.0 arrays. Differential gene expression was analyzed using a cutoff of 2-fold change and P-value of 0.01 and t-test in comparison to the control. Results Respiratory frequency during the baseline phase was 176 BPM in the control group, while was founded lower in the LPS and PAM group. Tidal volume (TVb) and minute ventilation (MVb) were also diminished in both groups compared to the control. The gasp latency, or time to recover after each anoxic episode, was prolonged impairing the autoresuscitation, especially, in the PAM treated group. Vocalization function was also affected by inflammation presence resulting in a reduced number of calls per min. A robust gene modulation in the brainstem bulk of PD5 pups was caused in both inflammatory insults. The enrichment analysis showed activation of T helper cells whereas CIBERSORT analysis indicated enrichment of Treg and B cells memory. The molecular pathways activated in neurons, astrocytes, and microglia were founded distinct and specific to the TLR ligand while astrocytes exhibited a lack of responsiveness caused by PAM. Conclusions We conclude that TRLs ligands activate specific neural mechanisms that will detrimentally affect ventilatory response and the ability to autoresuscitate via a central mechanism in the course of neonatal sepsis.

Toll‐like receptor 2 activation induces C–C chemokine receptor 2‐dependent natural killer cell recruitment to the peritoneum

Natural killer (NK) cells are innate effector cells with critical roles not only in tumor immunosurveillance and viral immunity, but also in bacterial and fungal infections. Toll‐like receptor 2 (TLR2) can be important in the early and sustained immune responses to pathogens and tumors through the induction of cytokines and chemokines that recruit and activate immune effector cells. We investigated the role of TLR2 activation in NK cell recruitment with a view to informing approaches to induce or regulate peritoneal NK cell responses therapeutically. Peritoneal injection of TLR2 activators, including peptidoglycan and the lipopeptides FSL‐1 and Pam3CSK4, resulted in NK cell recruitment after 16 h with increased NK cell numbers maintained for 48 h. TLR2 activators induced large amounts of CCR2 ligands, but much smaller amounts of CCR5 and CXCR3 ligands. Consistent with this observation, NK cell migration was abrogated in CCR2‐deficient mice after peritoneal FSL‐1 injection. Adoptive transfer of CCR2‐deficient NK cells prior to peritoneal FSL‐1 activation confirmed a cell‐intrinsic component of CCR2‐mediated NK cell migration. TLR2 activation did not induce an activated NK cell phenotype, but significant changes included an increase in the KLRG1+ subset and decreased NKG2D expression. Although not activated in vivo, peritoneal NK cells could be activated by interleukin (IL)‐12 and IL‐18 ex vivo to express CD69 and interferonγ. These data demonstrate that TLR2‐mediated immune activation is a potent inducer of NK cell recruitment via a CCR2‐dependent mechanism and that NK cells recruited by this mechanism can respond to additional signals to exert effector cell functions.

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates.

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.

PAM3 protects against DSS-induced colitis by altering the M2:M1 ratio

Inflammation of the gastrointestinal tract contributes to the development of inflammatory bowel disease (IBD). Human IBD is modeled by administering dextran sulfate sodium (DSS) to mice. In humans and mice, inflammatory M1 macrophages contribute to the progression of IBD whereas immunosuppressive M2 macrophages protect against colitis. The TLR2/1 agonist PAM3CSK4 (PAM3) induces human and murine monocytes to differentiate into immunosuppressive M2 macrophages, suggesting that PAM3 might be of benefit in the prevention/treatment of colitis. PAM3 was therefore administered to mice treated with DSS. As hypothesized, the number of M2 macrophages rose and disease severity decreased. The critical role of M2 macrophages in this process was established by transferring purified M2 macrophages from PAM3 treated control donors into DSS recipients and reducing colitis. These findings suggest that PAM3 may represent a novel approach to the treatment of human IBD.

Toll-Like Receptor Responsiveness of Peripheral Blood Mononuclear Cells in Young Women with Dysmenorrhea.

PurposeDysmenorrhea is a common disorder that substantially disrupts the lives of young women. To determine whether there is evidence of activation of the innate immune system in dysmenorrhea and whether the degree of activation may be used as a biomarker for pain, we compared the responsiveness of peripheral blood mononuclear cells (PBMCs) to toll-like receptor (TLR) 2 or 4 stimulation. We also investigated whether this effect is modulated by the use of the oral contraceptive pill (OC).Patients and MethodsFifty-six women aged 16–35 years, with either severe or minimal dysmenorrhea, and use or non-use of the OC, were enrolled. PBMCs were collected on two occasions in a single menstrual cycle: the menstrual phase and the mid-follicular phase. PBMCs were exposed to lipopolysaccharide (LPS), a TLR4 agonist, and PAM3CSK4 (PAM), a TLR2 agonist, and the resulting interleukin-1beta (IL–1β) output was determined. Statistical analysis compared the EC50 between groups as a measure of TLR responsiveness of PBMCs.ResultsThe key finding following LPS stimulation was a pain effect of dysmenorrhea (p=0.042) that was independent of use or non-use of OC, and independent of day of testing. Women with dysmenorrhea showed a large 2.15-fold (95% CI −4.69, −0.09) increase in IL–1β release when compared with pain-free participants across both days.ConclusionThis is the first study to demonstrate an ex vivo immune relationship in women with dysmenorrhea-related pelvic pain. It provides evidence for the potential of immune modulation as a novel pharmacological target for future drug development in the management of dysmenorrhea.

N-acetylcysteine inhibits bacterial lipopeptide-mediated neutrophil transmigration through the choroid plexus in the developing brain

The etiology of neurological impairments associated with prematurity and other perinatal complications often involves an infectious or pro-inflammatory component. The use of antioxidant molecules have proved useful to protect the neonatal brain from injury. The choroid plexuses-CSF system shapes the central nervous system response to inflammation at the adult stage, but little is known on the neuroimmune interactions that take place at the choroidal blood-CSF barrier during development. We previously described that peripheral administration to neonatal mice of the TLR2 ligand PAM3CSK4 (P3C), a prototypic Gram-positive bacterial lipopeptide, induces the migration of innate immune cells to the CSF. Here we showed in neonatal rats exposed to P3C that the migration of neutrophils into the CSF, which occurred through the choroid plexuses, is abolished following administration of the antioxidant drug N-acetylcysteine. Combining light sheet microscopy imaging of choroid plexus, a differentiated model of the blood-CSF barrier, and multiplex cytokine assays, we showed that the choroidal epithelium responds to the bacterial insult by a specific pattern of cytokine secretion, leading to a selective accumulation of neutrophils in the choroid plexus and to their trafficking into CSF. N-acetylcysteine acted by blocking neutrophil migration across both the endothelium of choroidal stromal vessels and the epithelium forming the blood-CSF barrier, without interfering with neutrophil blood count, neutrophil tropism for choroid plexus, and choroidal chemokine-driven chemotaxis. N-acetylcysteine reduced the injury induced by hypoxia-ischemia in P3C-sensitized neonatal rats. Overall, the data show that a double endothelial and epithelial check point controls the transchoroidal migration of neutrophils into the developing brain. They also point to the efficacy of N-acetylcysteine in reducing the deleterious effects of inflammation-associated perinatal injuries by a previously undescribed mechanism, i.e. the inhibition of innate immune cell migration across the choroid plexuses, without interfering with the systemic inflammatory response to infection.

Regulation of Immune Activation by Optical Control of TLR1/2 Heterodimerization.

The activation of toll-like receptors (TLRs) plays important roles in the immune response. The ability to control the activities of TLRs could be usable as a switch for immune response. Here we have rationally designed and synthesized a photoswitchable Pam3 CSK4 derivative-P10-to control the activation of TLR1/2. The ground-state trans-P10 was able to stimulate and activate antigen-presenting cells (APCs) by promoting TLR1/2 heterodimerization. However, cis-P10, derived from UV irradiation of trans-P10, reduced the activities of APCs by impeding the TLR1/2 heterodimerization. In the absence of UV radiation, the cis-P10 slowly returned to its ground trans state, restoring the activities of the APCs stimulation. Our results indicated that optical control of TLR1/2 heterodimerization mediated by the photoswitchable P10 offers the potential to regulate immune activation and inflammation.

Syntheses and Functional Studies of Self-Adjuvanting Anti-HER2 Cancer Vaccines.

The 9-mer peptide MFCH401 (N: 165-173: DTILWKDIF), which is located in the extracellular domain of HER2, has been predicted to be a novel epitope. Self-adjuvanting anti-HER2 vaccine constructs were designed and synthesized via covalently attaching MFCH401 or its linear tandem repeats (2×MFCH401, 3×MFCH401) to a lipopeptide Pam3 CSK4 via iterative condensation reaction. The in vivo results showed the Pam3 CSK4 -MFCH401 vaccine construct can induce higher antibody titers of IgG and IgM than those of other conjugates, and the analysis of changes in plasma cytokines level indicate the activation of Th1 cells and NK cells. In addition, the Pam3 CSK4 -MFCH401 vaccine conjugate induced a specific immune response to HER2-overexpressing human BT474 cells. Our data clearly indicated that MFCH401 is a promising epitope; moreover, its linear tandem repeats were unsuitable for anticancer vaccine design when conjugating with Pam3 CSK4 , which provided useful evidence for developing further anti-HER2 cancer vaccines.

Activation through toll-like receptor 2 on group 2 innate lymphoid cells can induce asthmatic characteristics.

Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM+HDM or vehicle and analysed. In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM+HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident inthe in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.

Liposomal Antitumor Vaccines Targeting Mucin 1 Elicit a Lipid-Dependent Immunodominant Response.

The tumor-associated antigen mucin 1 (MUC1) has been pursued as an attractive target for cancer immunotherapy, but the poor immunogenicity of the endogenous antigen hinders the development of vaccines capable of inducing effective anti-MUC1 immunodominant responses. Herein, we prepared synthetic anti-MUC1 vaccines in which the hydrophilic MUC1 antigen was N-terminally conjugated to one or two palmitoyl lipid chains (to form amphiphilic Pam-MUC1 or Pam2 -MUC1). These amphiphilic lipid-tailed MUC1 antigens were self-assembled into liposomes containing the NKT cell agonist αGalCer as an adjuvant. The lipid-conjugated antigens reshaped the physical and morphological properties of liposomal vaccines. Promising results showed that the anti-MUC1 IgG antibody titers induced by the Pam2 -MUC1 vaccine were more than 30- and 190-fold higher than those induced by the Pam-MUC1 vaccine and the MUC1 vaccine without lipid tails, respectively. Similarly, vaccines with the TLR1/2 agonist Pam3 CSK4 as an adjuvant also induced conjugated lipid-dependent immunological responses. Moreover, vaccines with the αGalCer adjuvant induced significantly higher titers of IgG antibodies than vaccines with the Pam3 CSK4 adjuvant. Therefore, the non-covalent assembly of the amphiphilic lipo-MUC1 antigen and the NKT cell agonist αGalCer as a glycolipid adjuvant represent a synthetically simple but immunologically effective approach for the development of anti-MUC1 cancer vaccines.

PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus

Abstract Systematic Lupus Erythematosus (SLE) is an autoimmune syndrome of unclear etiology. While T and B cell abnormalities contribute to disease pathogenesis, recent work suggests that inflammatory M1-like macrophages also play a role. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This raised the possibility of PAM3 being used to normalize the M1:M2 ratio in SLE. Consistent with that possibility, monocytes from lupus patients differentiated into M2-like macrophages when treated with PAM3 in vitro. Furthermore, lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Normalization of the M2 macrophage frequency was associated with delayed disease progression, decreased autoantibody and inflammatory cytokine synthesis, reduced proteinuria and prolonged survival in NZB x NZW F1 mice. The ability of PAM3 to bias monocyte differentiation in favor of immunosuppressive macrophages may represent a novel approach to the therapy of SLE.

Choroid plexus transcriptome and ultrastructure analysis reveals a TLR2-specific chemotaxis signature and cytoskeleton remodeling in leukocyte trafficking

Perinatal infection and inflammation are major risk factors for injury in the developing brain, however, underlying mechanisms are not fully understood. Leukocyte migration to the cerebrospinal fluid (CSF) and brain is a hallmark of many pathologies of the central nervous system including those in neonates. We previously reported that systemic activation of Toll-like receptor (TLR) 2, a major receptor for gram-positive bacteria, by agonist Pam3CSK4 (P3C) resulted in dramatic neutrophil and monocyte infiltration to the CSF and periventricular brain of neonatal mice, an effect that was absent by the TLR4 agonist, LPS. Here we first report that choroid plexus is a route of TLR2-mediated leukocyte infiltration to the CSF by performing flow cytometry and transmission electron microscopy (TEM) of the choroid plexus. Next, we exploited the striking discrepancy between P3C and LPS effects on cell migration to determine the pathways regulating leukocyte trafficking through the choroid plexus. We performed RNA sequencing on the choroid plexus after administration of P3C and LPS to postnatal day 8 mice. A cluster gene analysis revealed a TLR2-specific signature of chemotaxis represented by 80-fold increased expression of the gene Ccl3 and 1000-fold increased expression of the gene Cxcl2. Ingenuity pathway analysis (IPA) revealed TLR2-specific molecular signaling related to cytoskeleton organization (e.g. actin signaling) as well as inositol phospholipids biosynthesis and degradation. This included upregulation of genes such as Rac2 and Micall2. In support of IPA results, ultrastructural analysis by TEM revealed clefting and perforations in the basement membrane of the choroid plexus epithelial cells in P3C-treated mice. In summary, we show that the choroid plexus is a route of TLR2-mediated transmigration of neutrophils and monocytes to the developing brain, and reveal previously unrecognized mechanisms that includes a specific chemotaxis profile as well as pathways regulating cytoskeleton and basement membrane remodeling.

PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus

Systematic Lupus Erythematosus (SLE) is an autoimmune syndrome of unclear etiology. While T and B cell abnormalities contribute to disease pathogenesis, recent work suggests that inflammatory M1-like macrophages also play a role. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This raised the possibility of PAM3 being used to normalize the M1:M2 ratio in SLE. Consistent with that possibility, monocytes from lupus patients differentiated into M2-like macrophages when treated with PAM3 in vitro. Furthermore, lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Normalization of the M2 macrophage frequency was associated with delayed disease progression, decreased autoantibody and inflammatory cytokine synthesis, reduced proteinuria and prolonged survival in NZB x NZW F1 mice. The ability of PAM3 to bias monocyte differentiation in favor of immunosuppressive macrophages may represent a novel approach to the therapy of SLE.