Current treatments for chronic pain, such as inflammatory pain, neuropathic pain, and cancer pain are insufficient and cause severe side effects. Mounting evidence suggests that neuroinflammation in the peripheral and central nervous system (PNS and CNS) plays a pivotal role in the genesis and maintenance of chronic pain. Characteristic features of neuroinflammation in chronic pain conditions include infiltration of immune cells into the PNS [e.g., the sciatic nerve and dorsal root ganglion (DRG)], activation of glial cells such as microglia and astrocytes in the CNS (spinal cord and brain), and production and secretion of pro-inflammatory cytokines and chemokines [TNF, interleukin (IL)-1β, IL-6, CCL2, and CXCL1]. Recent studies suggest that bone marrow stem cells or bone marrow stromal cells (BMSCs) produce powerful analgesic effects in animal models of inflammatory pain, neuropathic pain, and cancer pain. We recently demonstrated that intrathecal injection of BMSCs resulted in a long-term relief of neuropathic pain for several weeks after peripheral nerve injury. Strikingly, this analgesic effect is mediated by the anti-inflammatory cytokine transforming growth factor beta secreted from BMSCs. Additionally, BMSCs exhibit potent modulation of neuroinflammation, by inhibiting monocyte infiltration, glial activation, and cytokine/chemokine production in the DRG and spinal cord. Thus, BMSCs control chronic pain by regulation of neuroinflammation in the PNS and CNS via paracrine signaling. In this review, we discuss the similar results from different laboratories of remarkable anti-nociceptive efficacy of BMSCs in animal and clinical studies. We also discuss the mechanisms by which BMSCs control neuroinflammation and chronic pain and how these cells specifically migrate to damaged tissues.

Mesenchymal stem cells (MSCs) show great therapeutic potential for the treatment of various immune mediated diseases, including Multiple Sclerosis (MS). Systemic administration of MSCs during experimental allergic encephalomyelitis (EAE), an animal model for MS, was shown to reduce the infiltration of T cells, B cells, and macrophages into the CNS. Whether endogenous MSCs are mobilized and potentially modulate the severity of disease is not known. Here we show that during the acute phase of EAE, MSCs numbers in the bone marrow were severely reduced, which restored to control levels during the progressive phase of the disease. The number of bone marrow MSCs inversely correlated with the number of both CD4 and CD8 T cells present in the bone marrow indicating a link between activated T cells and MSC mobilization. Analysis of CD70-transgenic mice, which have a constitutively activated immune system and elevated number of activated T cells in the bone marrow, showed severely reduced number of bone marrow MSCs. Transfer of T cells that were activated through their CD27 receptor reduced the number of bone marrow MSCs dependent on IFN-y. These data provide a mechanism by which MSCs can be mobilized from the bone marrow in order to contribute to tissue repair at a distant location.

Disseminated tumor cells in the bone marrow environment are the main cause of systemic metastasis after curative treatment for major solid tumors. However, the detailed biological processes of tumor biology in bone marrow have not been well defined in a real-time manner, because of a lack of a proper in vivo experimental model thereof. In this study, we established intravital imaging models of the bone marrow environment to enable real-time observation of cancer cells in the bone marrow. Using these novel imaging models of intact bone marrow and transplanted bone marrow of mice, respectively, via two-photon microscopy, we could first successfully track and analyze both the distribution and the phenotype of cancer cells in bone marrow of live mouse. Therefore, these novel in vivo imaging models for the bone marrow would provide a valuable tool to identify the biologic processes of cancer cells in a real-time manner in a live animal model.

In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)-based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1+CD14-MHC-II+CD172a-/lo CD1-CD163- DEC205+CD11R3 lo CD11R1+CD33+CD80/86+. They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4+CD8+ T cells, but were deficient in phagocyting inactivated Staphylococcus aureus (S. aureus). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1+CD14-MHC-II+C D172a+CD1+CD163-/lo DEC205 lo CD11R3+CD11R1+CD33+CD80/86+; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4+CD8-/CD4+CD8+ T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14+ population was identified with the phenotype CADM1+CD14+MHC-II+CD172a+ CD1+CD163+DEC205-CD11R3+CD11R1+CD33-/lo CD80/86+. They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14+ population was efficient in phagocyting S. aureus but showed less maturation upon TLR ligand stimulation than cDC1 or cDC2. The alternative methods of DC derivation including GM-CSF and/or IL-4 produced mostly CADM1- cells that did not fulfill the canonical phenotype of bona fide porcine DCs. Our study provides an exhaustive characterization of Flt3L-derived DCs with different methods that can help the in vitro study of the interaction of DCs with porcine-relevant pathogens.

Bone marrow plasma cells have been reported to represent a major source of IL-10; however, the impact of plasma cell derived IL-10 in that tissue remains poorly understood. We confirm in this study that even in the absence of acute immune reactions, mature plasma cells represent the dominant IL-10+ cell population in the bone marrow, and identify myeloid-lineage cells as a main local target for plasma cell derived IL-10. Using Vert-X IL-10 transcriptional reporter mice, we found that more than 50% of all IL-10+ cells in bone marrow were CD138+ plasma cells, while other IL-10+ B lineage cells were nearly absent in this organ. Accordingly, IL-10 was found in the supernatants of short-term cultures of FACS-sorted bone marrow plasma cells, confirming IL-10 secretion from these cells. IL-10+ bone marrow plasma cells showed a B220-/CD19-/MHCII low phenotype suggesting that these cells represent a mature differentiation stage. Approximately 5% of bone marrow leucocytes expressed the IL-10 receptor (IL-10R), most of them being CD115+/Ly6C+/CD11c- monocytes. Compared to littermate controls, young B lineage specific IL-10 KO mice showed increased numbers of CD115+ cells but normal populations of other myeloid cell types in bone marrow. However, at 7 months of age B lineage specific IL-10 KO mice exhibited increased populations of CD115+ myeloid and CD11c+ dendritic cells (DCs), and showed reduced F4/80 expression in this tissue; hence, indicating that bone marrow plasma cells modulate the differentiation of local myeloid lineage cells via IL-10, and that this effect increases with age. The effects of B cell/plasma cell derived IL-10 on the differentiation of CD115+, CD11c+, and F4/80+ myeloid cells were confirmed in co-culture experiments. Together, these data support the idea that IL-10 production is not limited to early plasma cell stages in peripheral tissues but is also an important feature of mature plasma cells in the bone marrow. Moreover, we provide evidence that already under homeostatic conditions in the absence of acute immune reactions, bone marrow plasma cells represent a non-redundant source for IL-10 that modulates local myeloid lineage differentiation. This is particularly relevant in older individuals.

Bone marrow (BM) plays an important role in the long-term maintenance of memory T cells. Yet, BM is found in numerous bones throughout the body, which are not equal in structure, as they differ in their ratio of cortical and trabecular bone. This implies that BM cells within different bones are subjected to different microenvironments, possibly leading to differences in their frequencies and function. To address this, we examined BM from murine tibia, femur, pelvis, sternum, radius, humerus, calvarium, and the vertebrae and analyzed the presence of effector memory (TEM), central memory (TCM), and naïve (TNV) CD8(+) T cells. During steady-state conditions, the frequency of the total CD8(+) T cell population was comparable between all bones. Interestingly, most CD8(+) T cells were located in the vertebrae, as it contained the highest amount of BM cells. Furthermore, the frequencies of TEM, TCM, and TNV cells were similar between all bones, with a majority of TNV cells. Additionally, CD8(+) T cells collected from different bones similarly expressed the key survival receptors IL-7Rα and IL-15Rβ. We also examined BM for memory CD8(+) T cells with a tissue-resident memory phenotype and observed that approximately half of all TEM cells expressed the retention marker CD69. Remarkably, in the memory phase of acute infection with the lymphocytic choriomeningitis virus (LCMV), we found a massive compositional change in the BM CD8(+) T cell population, as the TEM cells became the dominant subset at the cost of TNV cells. Analysis of Ki-67 expression established that these TEM cells were in a quiescent state. Finally, we detected higher frequencies of LCMV-specific CD8(+) T cells in BM compared to spleen and found that BM in its entirety contained fivefold more LCMV-specific CD8(+) T cells. In conclusion, although infection with LCMV caused a dramatic change in the BM CD8(+) T cell population, this did not result in noticeable differences between BM collected from different bones. Our findings suggest that in respect to CD8(+) T cells, BM harvested from a single bone is a fair reflection of the rest of the BM present in the murine body.

The contribution of bone-marrow derived cells (BMCs) to a newly formed beta-cell population in adults is controversial. Previous studies have only used models of bone marrow transplantation from sex-mismatched donors (or other models of genetic labeling) into recipient animals that had undergone irradiation. This approach suffers from the significant shortcoming of the off-target effects of irradiation. Partial pancreatic duct ligation (PDL) is a mouse model of acute pancreatitis with a modest increase in beta-cell number. However, the possibility that recruited BMCs in the inflamed pancreas may convert into beta-cells has not been examined. Here, we used an irradiation-free model to track the fate of the BMCs from the donor mice. A ROSA-mTmG red fluorescent mouse was surgically joined to an INS1Cre knock-in mouse by parabiosis to establish a mixed circulation. PDL was then performed in the INS1Cre mice 2 weeks after parabiosis, which was one week after establishment of the stable blood chimera. The contribution of red cells from ROSA-mTmG mice to beta-cells in INS1Cre mouse was evaluated based on red fluorescence, while cell fusion was evaluated by the presence of green fluorescence in beta-cells. We did not detect any red or green insulin+ cells in the INS1Cre mice, suggesting that there was no contribution of BMCs to the newly formed beta-cells, either by direct differentiation, or by cell fusion. Thus, the contribution of BMCs to beta-cells in the inflamed pancreas should be minimal, if any.

Tuberculosis (TB) remains a serious public health problem despite the great scientific advances in the recent decades. We have previously shown that aggressive forms of TB caused by hypervirulent strains of Mycobacterium tuberculosis and Mycobacterium bovis are attenuated in mice lacking the P2X7 receptor, an ion channel activated by extracellular ATP. Therefore, P2X7 receptor is a potential target for therapeutic intervention. In vitro, hypervirulent mycobacteria cause macrophage death by a P2X7-dependent mechanism that facilitates bacillus dissemination. However, as P2X7 receptor is expressed in both bone marrow (BM)-derived cells and lung structural cells, several cellular mechanisms can operate in vivo. To investigate whether the presence of P2X7 receptor in BM-derived cells contributes to TB severity, we generated chimeric mice by adoptive transfer of hematopoietic cells from C57BL/6 or P2X7-/- mice into CD45.1 irradiated mice. After infection with hypervirulent mycobacteria (MP287/03 strain of M. bovis), P2X7-/->CD45.1 mice recapitulated the TB resistance observed in P2X7-/- mice. These chimeric mice showed lower lung bacterial load and attenuated pneumonia compared to C57BL/6>CD45.1 mice. Lung necrosis and bacterial dissemination to the spleen and liver were also reduced in P2X7-/->CD45.1 mice compared to C57BL/6>CD45.1 mice. Furthermore, an immature-like myeloid cell population showing a Ly6Gint phenotype was observed in the lungs of infected C57BL/6 and C57BL/6>CD45.1 mice, whereas P2X7-/- and P2X7-/->CD45.1 mice showed a typical neutrophil (Ly6Ghi) population. This study clearly demonstrates that P2X7 receptor in BM-derived cells plays a critical role in the progression of severe TB.

Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34(+) progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates BM to release these effector cells in increased numbers. We hypothesize that mast cells (MCs) may play a predominant role in this process.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity.

Mast cells (MCs) are long-living multifunctional innate immune cells that originate from hematopoietic precursors and specifically differentiate in the destination tissue, e.g., skin, respiratory mucosa, intestine, where they mediate immune cell recruitment and antimicrobial defense. In vivo these tissues have characteristic physiological oxygen levels that are considerably lower than the atmospheric oxygen conditions (159 mmHg, 21% O2; 5% CO2) traditionally used to differentiate MCs and to study their functionality in vitro. Only little is known about the impact of physiological oxygen conditions on the differentiation process of MCs. This study aimed to characterize the differentiation of immature murine bone marrow-derived MCs under physioxia in vitro (7% O2; 53 mmHg; 5% CO2). Bone marrow-derived suspension cells were differentiated in the presence of interleukin-3 with continuous, non-invasive determination of the oxygen level using a Fibox4-PSt3 measurement system without technique-caused oxygen consumption. Trypan blue staining confirmed cellular viability during the specified period. Interestingly, MCs cultivated at 7% O2 showed a significantly delayed differentiation rate defined by CD117-positive cells, analyzed by flow cytometry, and reached >95% CD117 positive population at day 32 after isolation. Importantly, MCs differentiated under physioxia displayed a decreased transcript expression level of hif-1α and selected target genes vegf, il-6, and tnf-α, but an increase of foxo3 and vhl expression compared to MCs cultivated under normoxia. Moreover, the production of reactive oxygen species as well as the amount of intracellular stored histamine was significantly lower in MCs differentiated under low oxygen levels, which might have consequences for their function such as immunomodulation of other immune cells. These results show for the first time that physioxia substantially affect maturation and the properties of MCs and highlight the need to study their function under physiologically relevant oxygen conditions.

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types.

Integration of non-vascularized bone grafting and bone marrow aspirate infusion in transplantation may provide clinical benefit. Here we have incorporated bone fragment co-transplantation and bone marrow aspirate infusion (BF-BM) into living kidney transplantation (LKT). Twenty LKT recipients receiving bone fragments and bone marrow aspirates donated from their corresponding donors were enrolled into a retrospective study. A contemporaneous control group was formed of 38 out of 128 conventional LKT recipients, selected using propensity score matching by a 1:2 Greedy algorithm. Ultrasonography, contrast-enhanced ultrasonography (US/CEUS) and SPECT/CT showed that the co-transplanted bone fragments remained viable for 6 months, subsequently shrank, and finally degenerated 10 months post-transplantation. BF-BM resulted in earlier kidney recovery and more robust long-term kidney function. Throughout 5 years of follow-up, BF-BM had regulatory effects on dendritic cells (DCs), T helper (Th1/Th2) cells and regulatory T cells (Tregs). Both alloantigen-specific lymphocyte proliferation and panel reactive antibody levels were negative in all recipients with or without BF-BM. In addition, the BF-BM group experienced few complications during the 5-year follow-up (as did the donors)-this was not different from the controls. In conclusion, BF-BM is safe and benefits recipients by protecting the kidney and regulating the immune response.

Human lymphoid tissues harbor, in addition to CD56bright and CD56dim natural killer (NK) cells, a third NK cell population: CD69+CXCR6+ lymphoid tissue (lt)NK cells. The function and development of ltNK cells remain poorly understood. In this study, we performed RNA sequencing on the three NK cell populations derived from bone marrow (BM) and blood. In ltNK cells, 1,353 genes were differentially expressed compared to circulating NK cells. Several molecules involved in migration were downregulated in ltNK cells: S1PR1, SELPLG and CD62L. By flow cytometry we confirmed that the expression profile of adhesion molecules (CD49e-, CD29low, CD81high, CD62L-, CD11c-) and transcription factors (Eomeshigh, Tbetlow) of ltNK cells differed from their circulating counterparts. LtNK cells were characterized by enhanced expression of inhibitory receptors TIGIT and CD96 and low expression of DNAM1 and cytolytic molecules (GZMB, GZMH, GNLY). Their proliferative capacity was reduced compared to the circulating NK cells. By performing gene set enrichment analysis, we identified DUSP6 and EGR2 as potential regulators of the ltNK cell transcriptome. Remarkably, comparison of the ltNK cell transcriptome to the published human spleen-resident memory CD8+ T (Trm) cell transcriptome revealed an overlapping gene signature. Moreover, the phenotypic profile of ltNK cells resembled that of CD8+ Trm cells in BM. Together, we provide transcriptional and phenotypic data that clearly distinguish ltNK cells from both the CD56bright and CD56dim NK cells and substantiate the view that ltNK cells are tissue-resident cells, which are functionally restrained in killing and have low proliferative activity.

Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7-10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.

During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs) to the bone marrow (BM) in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b(+)T-bet(+) antigen-experienced CD4 T cells in the spleen. CD49b(+)T-bet(+) antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b(+)T-bet(+) precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.

Previous studies have shown that lipopolysaccharide (LPS)-activated bone marrow-derived dendritic cells (DClps) might induce tolerance in autoimmune and cancer models in vivo, whereas it remains unclear whether DClps could play a role in allergic disease model. Herein, we aimed to elucidate the potential effects of DClps on OVA-sensitized/challenged airway inflammation in a mouse model, which may help facilitate the application of specific tolerogenic dendritic cells (tolDC) in allergic asthma in the future.

Although controlled local inflammation is essential for adequate bone regeneration, several studies have shown that hyper-inflammatory conditions after major trauma are associated with impaired fracture healing. These hyper-inflammatory conditions include the trauma-induced systemic inflammatory response to major injury, open fractures, and significant injury to the surrounding soft tissues. The current literature suggests that increased or prolonged influx of neutrophils into the fracture hematoma may mediate impairment of bone regeneration after hyper-inflammatory conditions. The underlying mechanism remains unclear. We hypothesize that high neutrophil numbers inhibit synthesis of mineralized extracellular matrix (ECM) by bone marrow stromal cells (BMSCs). We therefore studied the effect of increasing concentrations of neutrophils on ECM synthesis by human BMSCs in vitro. Moreover, we determined how high neutrophil concentrations affect BMSC cell counts, as well as BMSC osteogenic activity determined by alkaline phosphatase (ALP) expression and ALP activity. Co-culture of BMSCs with neutrophils induced a 52% decrease in BMSC cell count (p < 0.01), a 64% decrease in the percentage of ALP+ cells (p < 0.001), a 28% decrease in total ALP activity (p < 0.01), and a significant decrease in the amount of mineralized ECM [38% decrease after 4 weeks (p < 0.05)]. Co-cultures with peripheral blood mononuclear cells and neutrophils within transwells did not induce a significant decrease in ALP activity. In conclusion, our data shows that a decreased amount of mineralized ECM became synthesized by BMSCs, when they were co-cultured with high neutrophil concentrations. Moreover, high neutrophil concentrations induced a decrease in BMSC cell counts and decreased ALP activity. Clarifying the underlying mechanism may contribute to development of therapies that augment bone regeneration or prevent impaired fracture healing after hyper-inflammatory conditions.

Molecular characterization of antibody immunity and human antibody discovery is mainly carried out using peripheral memory B cells, and occasionally plasmablasts, that express B cell receptors (BCRs) on their cell surface. Despite the importance of plasma cells (PCs) as the dominant source of circulating antibodies in serum, PCs are rarely utilized because they do not express surface BCRs and cannot be analyzed using antigen-based fluorescence-activated cell sorting. Here, we studied the antibodies encoded by the entire mature B cell populations, including PCs, and compared the antibody repertoires of bone marrow and spleen compartments elicited by immunization in a human immunoglobulin transgenic mouse strain. To circumvent prior technical limitations for analysis of plasma cells, we applied single-cell antibody heavy and light chain gene capture from the entire mature B cell repertoires followed by yeast display functional analysis using a cytokine as a model immunogen. We performed affinity-based sorting of antibody yeast display libraries and large-scale next-generation sequencing analyses to follow antibody lineage performance, with experimental validation of 76 monoclonal antibodies against the cytokine antigen that identified three antibodies with exquisite double-digit picomolar binding affinity. We observed that spleen B cell populations generated higher affinity antibodies compared to bone marrow PCs and that antigen-specific splenic B cells had higher average levels of somatic hypermutation. A degree of clonal overlap was also observed between bone marrow and spleen antibody repertoires, indicating common origins of certain clones across lymphoid compartments. These data demonstrate a new capacity to functionally analyze antigen-specific B cell populations of different lymphoid organs, including PCs, for high-affinity antibody discovery and detailed fundamental studies of antibody immunity.

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children.